High-throughput measurement of fibroblast rhythms reveals genetic heritability of circadian phenotypes in diversity outbred mice and their founder strains

Abstract

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders-80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits.

Figure 1

Representative bioluminescence recordings of ensemble Bmal1-dLuc rhythms in two individual fibroblast cultures derived from eight founder strains plotted by baseline-subtracted data.

Figure 2

Strain and sex differences of ensemble Bmal1-dLuc rhythms in primary fibroblast cultures. Quantified data for strain and sex differences in (A) period, (B) phase, (C) amplitude, (D) damping rate and (E) goodness of fit of Bmal1-dLuc rhythms. Bar graphs depict mean ± SEM. Bar graphs with fill pattern indicate the data from males. Asterisks indicate that circadian parameters are significantly (P < 0.05) different compared to C57BL/6J within the same sex. Hashtags indicate significant (P < 0.05) sex differences in period and phase of Bmal1-dLuc rhythms.

Figure 3

Distribution of circadian parameters in DO mice and two extreme founder strains, A/J and CAST/EiJ, along with C57BL/6J strain. (A) Period, (B) phase, (C) amplitude, (D) damping rate and (E) goodness of fit of ensemble Bmal1-dLuc rhythms in primary fibroblast cultures. Lines in bars indicate median and the whiskers indicate Min to Max.

Figure 4

Circadian rhythms of wheel-running activity in two extreme founder strains along with C57BL/6J strain. (A) Representative actograms of wheel-running activity in C57BL/6J, A/J and CAST/EiJ mice that were maintained under 12:12 light–dark cycles for entrainment and then released to constant darkness for free-run. (B) The period and (C) amplitude of the activity rhythms during constant darkness were quantified by periodogram analysis. Shade indicates light-off. Bar graphs depict mean ± SEM. Bar graphs with fill pattern indicate the data from males. Asterisks indicate significant differences (p < 0.05).