A 3D amphioxus brain atlas illuminates the blueprint of the ancestral chordate brain and highlights unique vertebrate innovations.

Comparative studies of vertebrate brains have elucidated the evolution of morphological diversity; yet, the fundamental chordate brain blueprint and the emergence of vertebrate-specific innovations remain elusive. To reconstruct the cellular and molecular origins of brain architecture in chordates, we combined single-nucleus RNA sequencing with spatial transcriptomics to generate a three-dimensional cell atlas of the central nervous system (CNS) in the early-branching chordate amphioxus. This atlas reveals a tripartite anterior-posterior organization comprising a rostral retinal/hypothalamic region, a di-mesencephalic domain, and a caudal hindbrain/spinal cord. Challenging previous views, we identify cell clusters resembling the vertebrate zona limitans intrathalamica and midbrain-hindbrain boundary regions that demarcate major brain compartments. Conversely, the absence of telencephalic marker expression in the amphioxus rostral forebrain suggests that the telencephalon is a vertebrate innovation. Collectively, our findings establish a spatial transcriptomic framework for the amphioxus CNS, providing crucial insight into the evolutionary origins of vertebrate brain complexity.