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Adv."],"published-print":{"date-parts":[[2026,7,3]]},"abstract":"<jats:p>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. 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