ERK and PKA signaling drive the evolutionary expansion of the cortex

The cerebral cortex, the anatomical foundation of human intelligence, underpins our advanced cognition and language. Deciphering the regulatory mechanisms driving its remarkable expansion is essential for unraveling the unique features that define the human brain and species. Here, we demonstrate that ERK and PKA signaling pathways coordinately maintain the neurogenic identity and lineage specification of cortical radial glia (RGs) by suppressing YAP and SHH signaling. Conversely, YAP signaling promotes the RG-ependymal glial cell lineage by inhibiting ERK, PKA, and SHH signaling, while SHH signaling facilitates the generation of cortical tripotential intermediate progenitor cells (Tri-IPCs) from RG by repressing ERK, PKA, and YAP signaling. Cortical Tri-IPCs exhibit a sequential differentiation potential, giving rise to cortical astrocytes, oligodendrocytes, and olfactory bulb interneurons. Importantly, we provide compelling evidence for the evolutionary conservation of these molecular mechanisms between mice and humans. Thus, ERK and PKA signaling in cortical RG establish a mutually reinforcing positive feedback loop, mediated through repressing gliogenic YAP and SHH signaling, which collectively promote human cortical expansion. This study identifies a unifying principle governing mammalian cortical neurogenesis, gliogenesis, expansion, and evolution. The whole mouse cortex was dissected under a microscope for single-cell RNA sequencing (scRNA-seq) analysis. Briefly, mouse embryos and postnatal brains were collected and immediately submerged in fresh ice-cold Hanks' Balanced Salt Solution (Gibco 12175-095). The cortex was then finely dissected, cut into small pieces, and dissociated into a single-cell suspension using a Papain Cell Dissociation Kit (Miltenyi Biotec, catalog no. 130-092-628) according to the manufacturer’s instructions. Single-cell RNA-seq libraries were generated using the Chromium droplet-based sequencing platform (10X Genomics) following the manufacturer’s protocol (manual document part number: CG00052 Rev C). After cellular quality filtering, the number of cells included in the dataset were as follows: E15.0 cortex: control (Map2k1/2 without Cre), 12,128 cells, 2,673 genes/cell; Emx1-Cre; Map2k1/2-dcko mice, 9,291 cells, 2,850 genes/cell. FlashTag labling at E15.5 and FACS at E16.5: control (SuperHippo without Emx1-Cre), 13,194 cells, 2,582 genes/cell; Emx1-Cre; SuperHippo mice, 14,641 cells, 2,616 genes/cell. FlashTag labling at E15.5 and FACS at E16.5: control (wild-type littermate) brains, yielding 14,800 cells, 1,504 genes/cell, hGFAP-Cre; SuperHippo brains, yielding 13,171 cells, 1,544 genes/cell. IUE (dnPKA) labling at E15.0 and FACS at E18.0: hGFAP-Cre; SmoF/F +dnPKA, 11,508 cells, 2,369 genes/cell. FlashTag labling at P0 and FACS P2: control (SmoF/F), 14,292 cells, 2,498 genes/cell; hGFAP-Cre; SmoF/F mice, 7,694 cells, 2,681 genes/cell. FlashTag labling at P0 and FACS P2: control (SuperHippo without hGFAP-Cre), 12,054 cells, 2,258 genes/cell; hGFAP-Cre; SuperHippo mice, 11,539 cells, 2,120 genes/cell. This dataset provides a comprehensive transcriptomic landscape of cortical cells at different developmental stages under various genetic and experimental conditions