The salamander dorsal pallium develops through conserved cortical neurogenesis principles including sequential generation of molecularly distinct superficial and deep glutamatergic layers from multipotent radial glia and Eomes+ intermediate progenitor cells, with temporally patterned progenitors expressing conserved gene programs that generate functionally similar projection neuron classes through divergent species-specific differentiation cascades, revealing that layered cortex predates amniotes and that mammalian neocortex evolution involved inversion of the ancestral outside-in neurogenesis gradient and diversification of neuronal differentiation programs rather than fundamental changes to progenitor biology.

The evolutionary origin of the cerebral cortex, a brain region typically defined by the presence of neuronal layers, remains elusive. While mammals and reptiles have a layered cortex, the amphibian pallium (dorsal telencephalon) is considered unlayered. Here, we identify distinct superficial- and deep-layer neurons in the salamander pallium. Like in mammals, these layers develop sequentially from multipotent radial glia and intermediate progenitor cells. Using lineage tracing and scRNA-seq, we reveal conserved principles of cortical development, including the temporal patterning of radial glia and the association of neuronal birthdate, molecular identity, and projection type. Our findings indicate that the mammalian cortex evolved from this ancestral developmental template through the inversion of the corticogenesis gradient, from outside-in to inside-out, and the diversification of neuronal differentiation programs.