GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE327nnn/GSE327901/primaryOK200OtherBranchiostoma floridaeOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE327901GEOGSEfalseA 3D Amphioxus Brain Atlas Illuminates the Blueprint of the Ancestral Chordate Brain and Highlights Unique Vertebrate InnovationsComparative 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-seq 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.2026/05/07GSE327901GSM966802336810327901Branchiostoma floridae