The rearing environment persistently modulates mouse phenotypes from the molecular to the behavioural level

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multi-center study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in five different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organization. We detected changes in chromatin organization in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings. Overall design: Nine-week-old, time-mated, primiparous pregnant C57BL/6JRj female mice in the last third of pregnancy, all from a single breeding population (Janvier Labs, Le Genest-Saint-Isle, France) were randomly allocated and transported to five independent rearing facilities (RF), where their offspring were born and reared until eight weeks of age. In order to assess the effect of the rearing environment independent of genotype and test conditions, both male and female offspring from all five RF were then transported to a single laboratory that was new for all mice to test for phenotypic differences. Specifically, we examined the extent and persistence of variation in the composition of the gut microbiota associated with the different rearing facilities and measured differences in phenotypic traits such as body weight, adrenal weight, neuroendocrine stress reactivity, and behavior. In addition, we assessed differences in neural chromatin accessibility to explore the potential biological basis of behavioral differences. The ATAC-seq analysis was performed on ventral hippocampi isolated from male mice at TP1 and TP2. For this, test-naïve male mice were used to avoid effects of testing on the chromatin profile. Five cages per rearing facility were selected randomly from all cages containing 3 male littermates after weaning. One mouse from the selected cages was sacrificed in the rearing facility at TP1 (total n=25, i.e. 5 biological replicates per rearing laboratory), while its test-naïve littermate was sacrificed in the testing laboratory at the end of the experiment (TP2; total n=25, i.e. 5 biological replicate per rearing facility). The analysis has been restricted to males because they showed the most pronounced phenotypic differences in behavior.