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Summary
The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. We here phenotype 43 BXD strains and observe they have large variation (~5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that ~40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence—long term feeding with high fat diet (HFD)—to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior.
Experiment design
All animals were communally housed by strain until phenotyping and fed a chow diet (CD; (Harlan 2018; 6% kCal/fat, 20% kCal/protein, 74% kCal/carbohydrate) throughout life after weaning. All BXD strains (BXD43–103) were originally sourced from the vivarium at the University of Tennessee Health Science Center (Memphis, TN, USA) then bred for two or more generations until progeny entered the phenotyping colony. For tissue collection on CD and HFD BXD cohorts, animals were sacrificed under isoflurane anesthesia and cardiac perfusion after an overnight fast. High fat diet treatment and two day isolation for the recording experiment were considered as having low impact on the animals’ welfare, while all other measurements and conditions were considered as having no negative impact. All research was approved by the Swiss cantonal veterinary authorities of Vaud under licenses 2257.0 and 2257.1.
About cases
42 strains of the BXD family (BXD43 – BXD103) and both parental strains (C57BL/6 and DBA/2) were born and raised at the EPFL in Switzerland prior to inclusion in this study. For each strain, 10 male animals were born and then separated evenly into two cohorts at 8 weeks of age: 5 animals per strain on a chow diet (6% kcal/fat, 20% protein, 74% carbohydrate) and 5 animals per strain on high fat diet (60% kcal/fat, 20% protein, 20% carbohydrate). Animals adjusted to the diet for 8 weeks, and then an intensive phenotyping metabolic phenotyping protocol was followed from 16 to 24 weeks of age (respiration, cold tolerance, oral glucose response, VO2max exercise, voluntary exercise, basal activity). Animals were communally housed until the last 5 weeks of the experiment, when the animals could rest. Animals were fasted overnight prior to sacrifice, which occurred between 9am and 11am after isoflurane anesthesia and perfusion. The quadriceps were the last tissue frozen in liquid nitrogen during the sacrifice, about 15 minutes after perfusion. Quadriceps were taken by cutting laterally at the knee.
About tissue
Brown adipose was later shattered in liquid nitrogen and about half was taken for each sample, although the size of the BAT varied dramatically. All ~5 animals per cohort had their RNA prepared, and then were pooled evenly (by µg of RNA) into a single RNA sample for each cohort. These pooled RNA samples were then purified using RNEasy, then sent out for array analysis. All RIN values were > 8.0.
About platform
All arrays were Affymetrix Mouse Gene 2.0 ST, prepared and run simultaneously in a single batch in October 2013 by Lorne Rose at the University of Tennessee Health Science Center.
About data processing
In general, the array data that we put in GeneNetwork has be logged and then z normalized, but instead of leaving the mean at 0 and the standard deviation of 1 unit, we shift up to a mean of 8 units and increase the spread by having an standard deviation of 2 units (what we call 2Z + 8 normalized data). This removes negative values from the tables.
Contributors
Williams EG, Mouchiroud L, Frochaux M, Pandey A, Andreux PA, Deplancke B, Auwerx J
Citation
Williams EG, Mouchiroud L, Frochaux M, Pandey A, Andreux PA, Deplancke B, Auwerx J, An evolutionarily conserved role for the aryl hydrocarbon receptor in the regulation of movement. PLoS Genetics, 2014.
Acknowledgment
The authors thank Cristina Cartoni, Sébastien Lamy, and Charles Thomas at the Center of Phenogenomics (CPG, EPFL) for help in establishing and phenotyping the BXD mice, Jesse Ingels who genotyped the congenic AHR line. We thank the Molecular Resource Center of Excellence at The University of Tennessee Health Science Center processing all microarrays. Thanks to Ian Duncan for supplying the mutant ss D. melanogaster lines. Discussions with Prof. Stephan Morgenthaler (EPFL) are also acknowledged.