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, I 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. I 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/MO25β (CAB39L) module, which supports LKB1-AMPK energy sensing. Together, these findings support a model in which dark-adapting rods bifurcate into a MYC-driven anabolic state and an RNA-processing-altered state, a balance that may be biased by an LKB1–AMPK-associated mechanism. This integrative computational framework provides a predictive model for rod photoreceptor adaptation, guiding future biochemical and molecular experiments to determine the molecular basis and drivers of this divergence.