Integrated ERK-PKA-YAP/TAZ-SHH Signaling Orchestrates Cortical Radial Glia Identity and Lineage Diversification

The signaling pathways governing cortical neurogenesis and gliogenesis in mice are well-defined, yet how they integrate to control the lineage progression of cortical radial glia (RGs) remains incompletely understood. Here, using mouse genetic models, it is demonstrated that ERK and PKA signaling cooperate to preserve the neurogenic capacity of cortical RGs by suppressing the gliogenic pathways YAP/TAZ and SHH. Specifically, YAP/TAZ signaling drives cortical RGs toward an ependymal fate, while SHH signaling promotes the generation of tripotential intermediate progenitor cells that produce cortical astrocytes and oligodendrocytes, and olfactory bulb interneurons. Reanalysis of published human cortical scRNA-seq data further revealed that the functional roles of these signaling pathways are conserved between mouse and human cortical RGs. Furthermore, human cortical outer RGs acquire dominant ERK/PKA signaling through a self-reinforcing loop that suppresses both YAP and SHH signaling, markedly enhancing self-renewal and extending neurogenesis. Thus, a tripartite network of ERK/PKA, YAP/TAZ, and SHH whose cross-repressive logic coordinates neurogenesis with gliogenesis and may underlie evolutionary expansion, providing a framework for understanding cortical development and evolution, is identified. Overall design: 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