(A) Hierarchical clustering of expression identified 12 distinct clusters, of which seven clusters (1, 4, 5, 6, 7, 9, 10) comprising of 394 genes, showed variation consistent with seasonality or Dehnel’s phenomenon. (B) Functional characterization of these genes using KEGG GO pathways found an enrichment of 14 pathways (p<0.05), many of which have been implicated in hypothalamic control of homeostatic maintenance, including, relaxin signaling, neuroactive ligand-receptor interaction, HIF-1 signaling, and PI3K-Akt signaling.

(A) Volcano plot of significant (padj<0.05) differentially expressed genes (colored) between phenotypic extremes of hypothalami size change (Stage 4 vs Stage 2) plotted by log fold-change. Vertical thresholds represent a 1.58 log fold-change in expression of (high effect; dark colors). (B) Pathway enrichment analysis identified 5 pathways to be enriched for differentially expressed genes: apoptosis (downregulated), spliceosome, Fanconi anemia, GABAergic synapse, and nicotine addiction (upregulated). (C) Patterning of gene expression across stages of Dehnel’s phenomenon for genes found in the apoptosis pathway (BCL2L1, CTSK, FOS, NFKBIA, NGF) and those associated with GABAergic synapses (CACNA1D, GABRE, GABRQ, GNB3). (D) Cell viability of Mustela putorious furo neural cell lines exposed to four treatments: scrambled BCL2L1 overexpression, BCL2L1 overexpression, heat with scrambled BCL2L1 overexpression, and heat with BCL2L1 overexpression. Heat significantly reduced the cell viability compared to controls but was not rescued by BCL2L1 overexpression.

(A) Heatmap and boxplots of genes with shrew-specific upregulation compared to other mammals associated with processes including calcium signaling, neurological functions, (B) blood brain barrier plasticity, and (C) food intake and leptin response.

Significant shrew-specific upregulation of genes associated with calcium signaling pathways, blood brain barrier plasticity, food intake and leptin response, and other related functions.

Boxplots of genes (CCDC22, FAM57B, GPR3) showing both evolutionary upregulation (A) in the shrew and differential expression between Stage 4 and Stage 2 individuals (B), which are implicated in the development and progression of human neurological and metabolic disorders.