Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.     

Genetic loci for diet-induced atherosclerotic lesions and plasma lipids in mice

Genetic factors independent of those affecting plasma lipid levels are a major contributor to risk for atherosclerosis in humans, yet the basis for these is poorly understood. This study examined plasma lipids and diet-induced atherosclerosis in 16-month-old female mice of strains C56BL/6J and DBA/2J. Mice of the parental strains, from recombinant inbred strains derived from these (BXD RI), and F₂ progeny were fed an atherogenic diet for 16 weeks, beginning at 1 year of age. This induced atherosclerotic lesion formation in both parental strains, accompanied by increased plasma LDL levels. However, individual BXD RI strains and the BXD F₂ mice demonstrated a range of atherosclerotic lesion formation that was not or at best weakly correlated with plasma lipid levels. Quantitative trait locus (QTL) analysis of the BXD F₂ mice identified a locus with significant linkage (lod 4.5) for aortic lesion size on Chromosome (Chr) 10 that was independent of plasma lipids. Other loci with suggestive or significant linkage for various plasma lipid measures were identified on Chr 2, 3, 4, 5, 6, 7, 11, and 17. In this intercross, the genes primarily influencing atherosclerosis are distinct from those controlling plasma lipid levels.     

Genetic maps of mouse chromosome 17 including 12 new anonymous DNA loci and 25 anchor loci.

An interspecific backcross between lab mice and Mus spretus was used to construct a multilocus map of Chromosome 17 consisting of 12 new anonymous loci and 9 anchor loci. In addition, 7 anonymous DNA loci were added to the Chr 17 map for the BXD strains. Although we were able to identify readily the most likely gene order in the interspecific backcross, we found no evidence for an unambiguous gene order using the BXD recombinant inbred strains. Comparison of the interspecific backcross map and the BXD RI strain map revealed evidence in the interspecific backcross for a longer total genetic length, enhanced recombination distal to H-2, a segment showing suppressed recombination, and strong interference.     

Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice

Little is known about the hormonal regulation of sexual behavior and about the pattern of expression in the brain of sex-steroid receptors in the BALB/c AnN strain of mice (Mus musculus). In this study, 8-week old male BALB/c AnN mice were castrated and the temporal course of decline of sexual behavior was studied, as well as the effects of daily treatment with either testosterone propionate (TP), estradiol benzoate (EB), or dihydrotestosterone propionate (PDHT). Castration resulted in rapid decline of sexual behavior, in both control or vehicle-treated mice. TP maintained full sexual behavior of castrated mice, while PDHT or EB did not have this effect. The expression of ER-alpha dropped nearly 50% after castration, and this pattern remained in TP or PDHT-treated mice, while EB increased the ER-alpha mRNA levels to almost the same values as in intact control mice. The same pattern was found when ER-beta mRNA levels were analyzed. The expression of the PR-A/B gene in the different brain regions in intact mice and after castration, or among the differently treated mice, showed significant differences between normal and castrated mice at all times in all brain regions studied, with the exception of the frontal cortex. Castration reduced the expression of AR by 10-fold, as compared to intact control mice, while TP or PDHT treatment returned its expression to the same levels as in intact control mice, in all brain areas studied. The changes are more prominent in POA-HIP than in HYP and CF. These results demonstrated a rapid decline of sexual behavior in this strain of mice after castration, and show that only TP was able to maintain male sexual behavior, with no correlation with the pattern of expression of sex hormone receptors in specific areas of the mouse brain.     

Multiple sexual ornaments signal heterozygosity in male blue tits

Higher individual genetic quality has been hypothesized to be associated with the expression of conspicuous ornaments. However, the relationship between multicomponent sexual signals and heterozygosity is poorly understood. In this study, we examined whether different ornaments, including song (repertoire size and bout length) and plumage coloration (yellow breast and blue crown), reflect individual genetic diversity in male blue tits (Aves: Cyanistes caeruleus). We estimated genetic diversity using 26 microsatellite markers that were classified as putatively functional (12 loci) and neutral (14 loci). We found that yellow breast carotenoid chroma, blue crown brightness, bout length and body condition were positively associated with heterozygosity at functional loci, but not with genetic diversity estimated at all typed loci or the subset of neutral markers. The lack of strong single‐locus effects and the presence of identity disequilibrium in our population suggest that the observed heterozygosity‐phenotype associations are driven by loci widely distributed across the genome. The predominant role of putatively functional loci evidences that the expression of secondary sexual characters is more tightly reflected by heterozygosity at genomic regions containing coding genes that are being actively expressed, a fact that may make ornamental traits more reliable indicators of the genetic quality of individuals. Overall, this study shows that multiple secondary sexual characters reflect male genetic diversity and lends support to the good‐genes‐as‐heterozygosity hypothesis. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 362–375.     

Independent quantitative trait loci influence ventral and dorsal hippocampal volume in recombinant inbred strains of mice

Anatomical and functional studies support segregation of the hippocampus into ventral and dorsal components along its septotemporal axis. However, it is unknown whether the development of these two components of the hippocampus is influenced by common or separate genetic factors. In this study, we used recombinant inbred strains of mice to determine whether the same or different quantitative trait loci (QTL) influence ventral and dorsal hippocampal volume. Using two sets of strains of recombinant inbred mice (BXD and AXB/BXA), we identified separate QTLs for ventral and dorsal hippocampal volume. In BXD mice, suggestive QTLs for ventral hippocampus were identified on chromosomes 2, 8 and 13, and a significant QTL for dorsal hippocampal volume was identified on chromosome 15. There was also a suggestive QTL for dorsal hippocampal volume on chromosome 13. In AXB/BXA mice, there were no significant or suggestive QTLs for ventral hippocampal volume, but a significant QTL for dorsal hippocampus was identified on chromosome 5. These findings suggest that the development of the ventral and dorsal components of the hippocampus is influenced by separate genetic loci.     

Genotyping new BXD recombinant inbred mouse strains and comparison of BXD and consensus maps

Nine additional BXD recombinant inbred (RI) strains have been developed from the F₂ cross of C57BL/6J and DBA/2J mouse strains. A tenth line stopped breeding in the F₁₂ generation. F₂₀ generation breeding pairs from the nine surviving strains and an F₁₂ pair from the extinct line were genotyped at 319 genetic markers (primarily microsatellites) spanning most of the genome. Where typing data were lacking, the established set of 26 BXD strains also were genotyped at these same loci. The availability of these additional nine strains enhances the value of the BXD RI set for analysis of complex phenotypic traits. The proportion of loci still segregating at the F₂₀ generation was found to closely approximate expectation, suggesting that selection favoring the retention of heterozygosity is not a strong factor. However, the number of crossovers between adjacent markers was frequently less than predicted from consensus map distances. A significant deficiency of recombinants was observed on Chrs 3, 4, 14, and X. On Chr 14, the estimated cumulative BXD map distance between the most proximal and distal markers was only 30.2 cM, compared with a distance of 60.0 cM in the consensus map. On the X Chr, the estimated and predicted cumulative distances were 38.8 and 69.5 cM, respectively. Over all chromosomes, the BXD RI map is 14.5% shorter than predicted from the consensus map. It is suggested that distances in some of the consensus maps are inflated. Alternatively, recombinant genotypes could be selected against during inbreeding owing to allelic interactions affecting fitness. The latter interpretation implies that relatively strong intrachromosomal epistasis is common. Received: 2 October 1998 / Accepted: 15 December 1998     

Delayed and Transient Increase of Adult Hippocampal Neurogenesis by Physical Exercise in DBA/2 Mice

This study builds on the findings that physical activity, such as wheel running in mice, enhances cell proliferation and neurogenesis in the adult hippocampus of the common mouse strain C57BL/6, and that the baseline level of neurogenesis varies by strain, being considerably lower in DBA/2. Because C57BL/6 and DBA/2 are important as the parental strains of the BXD recombinant inbred cross which allows the detection of genetic loci regulating phenotypes such as adult neurogenesis, we performed the current study to investigate the gene x environment interactions regulating neurogenesis. At equal distances and times run DBA/2J mice lacked the acute increase in precursor cell proliferation known from C57BL/6. In DBA/2J proliferation even negatively correlated with the distance run. This was neither due to a stress response (to running itself or single housing) nor differences in estrous cycle. DBA/2 animals exhibited a delayed and weaker pro-neurogenic response with a significant increase in numbers of proliferating cells first detectable after more than a week of wheel running. The proliferative response to running was transient in both strains, the effect being undetectable by 6 weeks. There was also a small transient increase in the production of new neurons in DBA/2J, although these extra cells did not survive. These findings indicate that the comparison between C57BL/6 and DBA/2, and by extension the BXD genetic reference population derived from these strains, should provide a powerful tool for uncovering the complex network of modifier genes affecting the activity-dependent regulation of adult hippocampal neurogenesis. More generally, our findings also describe how the external physical environment interacts with the internal genetic environment to produce different responses to the same behavioral stimuli.     

Mouse DNA ''fingerprints'': analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains.

Human minisatellite probes cross-hybridize to mouse DNA and detect multiple variable loci. The resulting DNA "fingerprints" vary substantially between inbred strains but relatively little within an inbred strain. By studying the segregation of variable DNA fragments in BXD recombinant inbred strains of mice, at least 13 hypervariable loci were defined, 8 of which could be regionally assigned to mouse chromosomes. The assigned loci are autosomal, dispersed and not preferentially associated with centromeres or telomeres. One of these minisatellites is complex, with alleles 90 kb or more long and with internal restriction endonuclease cleavage sites which produce a minisatellite "haplotype" of multiple cosegregating fragments. In addition, one locus shows extreme germ-line instability and should provide a useful system for studying more directly the rates and processes of allelic variation of minisatellites.     

Spontaneous mutations in recombinant inbred mice: mutant toll-like receptor 4 (Tlr4) in BXD29 mice

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.     

Genetic control of novel food preference in mice

In food preference studies, mammals are often categorized as being either neophilic or neophobic, i.e., preferring or disliking a novel-tasting food. To date, the genetic factors influencing novel food preference have not been elucidated. To understand this phenomenon, we investigated the genetics of food preference in eight inbred strains of mice. We gave them plain-, cinnamon-, or cocoa-flavored powdered food on day 0 for 45 min and then a choice of cinnamon- or cocoa-flavored food 14 days later. We determined their preference for novel versus already-experienced flavored food and found that some inbred strains chose the food that they had been given previously, while others chose a different food. In particular, the DBA/2 strain chose more cinnamon-flavored food after it was pre-exposed to cinnamon, while the B6 strain chose less cinnamon-flavored food after this initial exposure. The BXD recombinant inbred (RI) strain set was then used to map quantitative trait loci (QTLs) that influence this novel food preference. One of these QTLs was found to map to the distal end of Chromosome (Chr) 8.     

Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.     

Systems genetic analysis of hippocampal neuroanatomy and spatial learning in mice

Variation in hippocampal neuroanatomy correlates well with spatial learning ability in mice. Here, we have studied both hippocampal neuroanatomy and behavior in 53 isogenic BXD recombinant strains derived from C57BL/6J and DBA/2J parents. A combination of experimental, neuroinformatic and systems genetics methods was used to test the genetic bases of variation and covariation among traits. Data were collected on seven hippocampal subregions in CA3 and CA4 after testing spatial memory in an eight‐arm radial maze task. Quantitative trait loci were identified for hippocampal structure, including the areas of the intra‐ and infrapyramidal mossy fibers (IIPMFs), stratum radiatum and stratum pyramidale, and for a spatial learning parameter, error rate. We identified multiple loci and gene variants linked to either structural differences or behavior. Gpc4 and Tenm2 are strong candidate genes that may modulate IIPMF areas. Analysis of gene expression networks and trait correlations highlight several processes influencing morphometrical variation and spatial learning.     

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.     

Identification of Hepatocarcinogen-Resistance Genes in Dba/2 Mice

Male DBA/2J mice are ~20-fold more susceptible than male C57BL/6J mice to hepatocarcinogenesis induced by perinatal treatment with N,N-diethylnitrosamine (DEN). In order to elucidate the genetic control of hepatocarcinogenesis in DBA/2J mice, male BXD recombinant inbred, D2B6F(1) X B6 backcross, and D2B6F(2) intercross mice were treated at 12 days of age with DEN and liver tumors were enumerated at 32 weeks. Interestingly, the distribution of mean tumor multiplicities among BXD recombinant inbred strains indicated that hepatocarcinogen-sensitive DBA/2 mice carry multiple genes with opposing effects on the susceptibility to liver tumor induction. By analyzing D2B6F(1) X B6 backcross and D2B6F(2) intercross mice for their liver tumor multiplicity phenotypes and for their genotypes at simple sequence repeat marker loci, we mapped two resistance genes carried by DBA/2J mice, designated Hcr1 and -2, to chromosomes 4 and 10, respectively. Hcr1 and Hcr2 resolved the genetic variance in the backcross population well, indicating that these resistance loci are the major determinants of the variance in the backcross population. Although our collection of 100 simple sequence repeat markers allowed linkage analysis for ~95% of the genome, we failed to map any sensitivity alleles for DBA/2J mice. Thus, it is likely that the susceptibility of DBA/2J mice is the consequence of the combined effects of multiple sensitivity loci.     

The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells.

The resistance of mice to lethal infection by murine CMV (MCMV) is under complex host genetic control with contributions from both H-2 and non-H-2 genes. We have previously shown that an autosomal, non-MHC encoded gene, Cmv-1, controls MCMV replication in the spleen. We have investigated the mechanism by which the Cmv-1 resistance gene confers protection against MCMV infection. Using H-2 compatible irradiation bone marrow chimeras, the enhanced resistance to MCMV infection that is associated with the Cmv-1l allele in the C57BL background was shown to be mediated by an irradiation-sensitive bone marrow-derived cell population, or a factor produced by these cells. The lack of correlation between serum IFN titers and the strain distribution pattern of Cmv-1 in CXB recombinant inbred mouse strains suggests that IFN does not mediate resistance conferred by this gene. Similarly, the lack of effect of in vivo depletion of mature CD4+ and CD8+ T cells on virus replication in C57BL/6J mice indicates that T cells are unlikely to be involved. In contrast, in vivo depletion of NK cells by injection of the anti-NK1.1 mAb PK136 abrogated restricted splenic virus replication in C57BL/6J----BALB.B chimeric mice and in the Cmv-1l CXB strains. These data indicate that the effect of the Cmv-1 gene is mediated by NK cells. The significant augmentation in NK cell activity after MCMV infection of the susceptible Cmv-1h strains (BALB/cBy), CXBG/By, CXBH/By, CXBI/By, and CXBK/By) indicates the existence in these mice of NK cells that are functionally and phenotypically distinct from those in Cmv-1l strains. NK cells present in the Cmv-1h strains are unable to restrict efficiently splenic MCMV replication in vivo, possibly due to a lack of specificity for virus-infected target cells. Finally, flow cytometric analysis of NK1-1 expression in CXB and BXD RI mice together with MCMV replication studies in the BXD RI strains indicate that Cmv-1 is closely linked to NK1.1 and other loci that reside on a distal segment of murine chromosome 6 in a region that has recently been defined as the natural killer complex.     

Systems genetics analysis of iron regulation in the brain

Iron imbalances in the brain, including excess accumulation and deficiency, are associated with neurological disease and dysfunction; yet, their origins are poorly understood. Using systems genetics analysis, we have learned that large individual differences exist in brain iron concentrations, even in the absence of neurological disease. Much of the individual differences can be tied to the genetic makeup of the individual. This genetic-based differential regulation can be modeled in genetic reference populations of rodents. The work in our laboratory centers on iron regulation in the brain and our animal model consists of 25 BXD/Ty recombinant inbred mouse strains. By studying naturally occurring variation in iron phenotypes, such as tissue iron concentration, we can tie that variability to one or more genes by way of quantitative trait loci (QTL) analysis. Moreover, we can conduct genetic correlation analyses between our phenotypes and others previously measured in the BXD/Ty strains. We have observed several suggestive QTL related to ventral midbrain iron content, including one on chromosome 17 that contains btbd9, a gene that in humans has been associated with restless legs syndrome and serum ferritin. We have also observed gene expression correlations with ventral midbrain iron, including btbd9 expression and dopamine receptor expression. In addition, we have observed significant correlations between ventral midbrain iron content and dopamine-related phenotypes. The following is a discussion of iron regulation in the brain and the contributions a systems genetics approach can make toward understanding the genetic underpinnings and relation to neurological disease.     

Quantitative trait loci linked to thalamus and cortex gray matter volumes in BXD recombinant inbred mice

To investigate whether there are separate or shared genetic influences on the development of the thalamus and cerebral cortex, we identified quantitative trait loci (QTLs) for relevant structural volumes in BXD recombinant inbred (RI) strains of mice. In 34 BXD RI strains and two parental strains (C57BL/6J and DBA/2J), we measured the volumes of the entire thalamus and cortex gray matter using point counting and Cavalieri's rule. Heritability was calculated using analysis of variance (ANOVA), and QTL analysis was carried out using WebQTL (http://www.genenetwork.org). The heritability of thalamus volume was 36%, and three suggestive QTLs for thalamus volume were identified on chromosomes 10, 11 and 16. The heritability of cortical gray matter was 43%, and four suggestive QTLs for cortex gray matter volume were identified on chromosomes 2, 8, 16 and 19. The genetic correlation between thalamus and cortex gray matter volumes was 0.64. Also, a single QTL on chromosome 16 (D16Mit100) was identified for thalamus volume, cortex gray matter volume and Morris water maze search-time preference (r=0.71). These results suggest that there are separate and shared genetic influences on the development of the thalamus and cerebral cortex.