We performed PSEA, a novel analytical tool for the visualization of identified and biologically related gene sets with temporally coordinated expression
51 to examine functional pathways in the corneas of NC and HFI mice that peaked at specific timepoints. The largest number of significant phase clusters (Kuiper
Q value < 0.01) in the corneas of both groups was observed in the light and dark cycles (ZT0 to ZT6 and ZT18-ZT24 in NC mice; ZT0 to ZT9 and ZT15-ZT21 in HFI mice) (
Figs. 6D and E). In total, 18 significantly enriched functional pathways were identified in the corneas of NC mice, grouped into four categories as follows: (1) genetic information processing: proteasome, ribosome; (2) cellular processes: cell adhesion molecules cams, lysosome, oocyte meiosis; ribosome, MAPK signaling pathway, spliceosome; (3) disease-associated pathways: pathways in cancer, Parkinson's disease, Alzheimer's disease, Huntington's disease, pancreatic cancer, small cell lung cancer, chronic myeloid leukemia, oocyte meiosis; and (4) organismal systems: chemokine signaling pathway, insulin signaling pathway, cardiac muscle contraction (
Fig. 6D,
Supplementary Table S3). In total, 23 significantly enriched functional pathways were identified in the corneas of HFI mice, grouped as follows: (1) metabolic pathways: oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, pyrimidine metabolism, purine metabolism, and glycerophospholipid metabolism; (2) cellular processes, including lysosome, focal adhesion, tight junction, endocytosis, and cell cycle; (3) genetic information processing: spliceosome, DNA replication, and proteasome; (4) organismal systems: extracellular matrix receptor interaction, JAK–STAT signaling pathway, leukocyte transendothelial migration, and FcγR-mediated phagocytosis; and (5) disease-associated pathways, including pathways in cancer (NC group vs. fructose group, ZT18.21/ZT2.26), Parkinson's disease (v ZT2.27/ZT8.47), Alzheimer's disease (ZT1.89/ZT6.85), and Huntington's disease (ZT2.07/ZT7.44) (
Fig. 6E,
Supplementary Table S4). Each disease-associated pathway was activated in NC and HFI mice but at different ZTs. Collectively, these results indicated that HFI temporally altered the quality and distribution of phase set–enriched signaling pathways.