Recent breakthroughs in scientific research technologies have enriched explanations for the origin of highly intelligent organisms. However, current research predominantly focuses on vertebrates, especially primates. In nature, one group has evolved advanced intelligence through a distinct evolutionary pathway: the coleoid cephalopods (Mollusca: Cephalopoda). Here, we used the bobtail squid Euprymna berryi (Coleoidea) to investigate nervous system evolution through single-cell analysis of brain tissue.

    A-to-I RNA editing occurs at remarkably high levels in coleoids but is minimal in other mollusks. While existing studies primarily examine bulk RNA, single-cell resolution of RNA editing remains largely unexplored. Our study leverages single-cell sequencing of E. berryi to characterize cell types in the mature brain and compare RNA editing levels across distinct neural populations.

To further elucidate developmental mechanisms in coleoids, we profiled embryonic bobtail squid using single-cell RNA sequencing. Integration with pseudotime analysis reconstructed complete developmental trajectories for neuronal and glial lineages.

Beyond development, we explored circadian regulation—a hallmark of E. berryi behavior. Significant diel variations in gene expression and RNA editing sites were observed. We performed RNA sequencing across 12 time points over a 48-hour cycle, revealing robust oscillations in transcriptional and epitranscriptomic activity.