Pseudotime trajectory analysis reveals divergent rod photoreceptor states during dark adaptation

Rod photoreceptors face a high ATP demand in darkness, yet the molecular programs supporting dark adaptation remain difficult to dissect in vivo. Here, we re-analyzed publicly available murine retinal single-cell RNA-seq data and reconstructed rod-state dynamics across the light-to-dark transition using pseudotime trajectory inference. We found that dark-adapting rods diverge from a common state into two distinct lineages. Lineage 1 is characterized by elevated MYC-driven anabolic programs alongside increased reactive oxygen species (ROS) response and unfolded protein response (UPR)/ER stress signatures. In contrast, Lineage 2 exhibits an altered RNA-processing state with a markedly higher unspliced RNA fraction. Intronic motif analysis of Lineage 2 identified enriched binding sites for splicing-associated RNA-binding proteins linked to core spliceosome components and ATP-dependent helicases. Furthermore, Lineage 2 relatively preserves the STRADA/MO25{beta} (CAB39L) module, which supports LKB1-AMPK energy sensing, whereas candidate upstream regulator analysis implicated miRNA-related networks in tuning this energy-sensing module. Together, these findings suggest that MYC driven anabolism, RNA splicing regulation, and LKB1-AMPK energy sensing, potentially modulated by miRNA networks, coordinately govern dark adaptation. Integrating biochemical readouts with RNA-processing assays will be essential to determine the molecular basis and drivers of this divergence, and whether the two lineages represent reversible adaptive states or stable endpoints. HighlightsPseudotime analysis reveals two distinct rod lineages during dark adaptation. Lineage 1 shows MYC/mTORC1-linked anabolic programs and ER-stress signatures. Lineage 2 shows increased unspliced RNA in long, vision-related transcripts. mTORC1/AMPK energy-sensing signatures correlate with lineage divergence.