Distinct neurogenic pathways shape the diversification and mosaic organization of cortical output channels

A hallmark of the mammalian cerebral cortex is its vast and diverse efferent projections across cortical areas mediated by extratelencephalic (ET) neurons that broadcast to myriad subcortical regions. ET neurons are generated from two fundamentally distinct neurogenic pathways: direct neurogenesis (dNG-ET^d) from radial glia progenitors and indirect neurogenesis (iNG-ETⁱ) from intermediate progenitors, but the contribution of ET^d and ETⁱ to the organization of cortical output channels is unknown. Leveraging a novel lineage-based genetic strategy enabling differential viral access to ET^d and ETⁱ in the same mouse, we show that iNG-ETⁱ massively amplifies and diversifies dNG-ET^d across the cortex. While ET^d projections are largely restricted to the forebrain and midbrain structures, ETⁱ greatly amplifies and diversifies these projections and overwhelmingly dominates the innervation of hindbrain and spinal cord. This is exemplified in an area-specific pattern by ETⁱ dominance to brainstem and spinal action diversification and execution centers from motor areas, to major sensory processing stations along lemniscal pathways from sensory areas, and to pallial, hypothalamic and neuromodulatory structures from high order areas. Corticofugal subpopulations in multiple areas are derived from only ETⁱ, indicating the generation of novel projection types by iNG over dNG. Furthermore, area-specific ETⁱ, but not ET^d, spinal projection pattern is sculpted from the massive pruning of an early cortex-wide population during postnatal development. Together, these results uncover that two foundational neurogenic pathways with distinct evolutionary history differentially shape the area-specific diversification and mosaic organization of cortical output channels.