Comparative transcriptomics of the garden dormouse hypothalamus during hibernation

Abstract

Torpor or heterothermy is an energy-saving mechanism used by endotherms to overcome harsh environmental conditions. During winter, the garden dormouse (Eliomys quercinus) hibernates with multiday torpor bouts and body temperatures of a few degrees Celsius, interrupted by brief euthermic phases. This study investigates gene expression within the hypothalamus, the key brain area controlling energy balance, adding information on differential gene expression potentially relevant to orchestrate torpor. A de novo assembled transcriptome of the hypothalamus was generated from garden dormice hibernating under constant darkness without food and water at 5 °C. Samples were collected during early torpor, late torpor, and interbout arousal. During early torpor, 765 genes were differentially expressed as compared with interbout arousal. Twenty-seven pathways were over-represented, including pathways related to hemostasis, extracellular matrix organization, and signaling of small molecules. Only 82 genes were found to be differentially expressed between early and late torpor, and no pathways were over-represented. During late torpor, 924 genes were differentially expressed relative to interbout arousal. Despite the high number of differentially expressed genes, only 10 pathways were over-represented. Of these, eight were also observed to be over-represented when comparing early torpor and interbout arousal. Our results are largely consistent with previous findings in other heterotherms. The addition of a transcriptome of a novel species may help to identify species-specific and overarching torpor mechanisms through future species comparisons.

Keywords: Eliomys quercinus; RNA-Seq; differential gene expression; euthermia; hibernation; metabolism.

Fig. 1

Sampling scheme. Core body temperature (T _b) of one dormouse over the course of 2 weeks during hibernation in constant darkness without food or water at 5 °C ambient temperature. Animals were sacrificed in early torpor (ET, 1 or 2 days in torpor, n = 4), late torpor (LT, 9 or 10 days in torpor, n = 3), or interbout arousal (IBA, 2 of 4 h in interbout arousal, n = 4). Radiotelemetry data were used previously [19, 39]. Transcriptome data of sampling groups were compared pairwise (arrows; ET vs LT, ET vs IBA, LT vs IBA).

Fig. 2

Principal component analysis. Distance between samples of this study (dots; ET: black, LT: blue, IBA: orange) by their respective overall gene expression in the first dimensions (PC1: 27% variance, PC2: 13% variance). Data basis is ET (n = 4), LT (n = 3), and IBA (n = 4).

Fig. 3

Heatmap and hierarchical clustering of genes differentially expressed in at least one pairwise group comparison. Gene expression (gradient from blue, low, to red, high expression) of 1351 DEGs (y‐axis) with Padj < 0.05 in at least one pairwise group comparison is plotted for each of the 11 samples (x‐axis). Genes are listed in File S1.

Fig. 4

State‐specific genes. Number of DEGs with Padj < 0.05 and any fold change shared between or specific for the pairwise group comparisons ET vs IBA, ET vs LT, and LT vs IBA and their intersections. Genes per intersection are listed in File S1.

Fig. 5

Volcano plots. Differential gene expression for the pairwise group comparisons ET vs IBA (A), ET vs LT (B), and LT vs IBA (C). All annotated genes (dots) are plotted based on fold change (x‐axis) and significance (y‐axis). DEGs (Padj < 0.05) are shown above the horizontal solid line (−log₁₀(0.05) = 1.3), while DEGs with a high significance (Padj < 0.001) are shown above the dotted line (−log₁₀(0.001) = 3.0). Genes with decreased expression log₂(FC) < 0 are shown within the yellow area, while those with a decreased expression of log₂(FC) < −1 are shown within the dark yellow area. Genes with increased expression log₂(FC) > 0 are shown within the green area, while those with increased expression of log₂(FC) > 1 are shown within the dark green area. The numbers of DEGs per area are indicated, while the number in the right upper corner states the total number of DEGs. Data basis is ET (n = 4), LT (n = 3), and IBA (n = 4). Genes are listed in File S1.

Fig. 6

Indicator genes differentially expressed in at least one pairwise group comparison. To visualize differences between samples within a group and differences between groups, the normalized count was plotted for all differentially expressed indicator genes with Padj < 0.05 in at least one pairwise group comparison. Data basis is ET (n = 4), LT (n = 3), and IBA (n = 4). Significant differences between groups are indicated with brackets. Associated Padj‐values are listed in Table 3. Results of all 68 indicator genes screened for are provided in File S3.