3D single-cell transcriptomics in lamprey brain

The lamprey, a jawless vertebrate with a primitive yet complex brain, occupies a crucial position for understanding vertebrate brain evolution. Using spatial transcriptomics and single-nucleus RNA sequencing, we generated a 3D molecular atlas of the entire lamprey brain. We identified 209 transcriptionally and spatially distinct cell clusters spanning 14 anatomically defined regions, including diverse neuronal, glial, and ependymal populations. The spatial organization of these regions, such as pallial subdivisions and the laminated olfactory bulb, mirrors patterns observed in jawed vertebrates, indicating that regional brain partitioning emerged in the last common vertebrate ancestor. Cross-species analyses revealed a continuum of neuronal specialization across vertebrate evolution. Lamprey neurons exhibit mixed molecular signatures, co-expressing excitatory and inhibitory markers, and likely represent ancestral prototypes of the specialized glutamatergic and GABAergic neurons found in jawed vertebrates. We further identified homologs of Purkinje and granule cells, suggesting a cerebellar-like architecture predates the emergence of the jawed vertebrate cerebellum. These findings illuminate the ancestral cellular architecture of the vertebrate brain and reveal early innovations that underpin its later diversification.