ERK signaling expands mammalian cortical radial glial cells and extends the neurogenic period

The molecular basis for cortical expansion during evolution remains largely unknown. Here, we report that fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signaling promotes the self-renewal and expansion of cortical radial glial (RG) cells. Furthermore, FGF-ERK signaling induces bone morphogenic protein 7 (Bmp7) expression in cortical RG cells, which increases the length of the neurogenic period. We demonstrate that ERK signaling and Sonic Hedgehog (SHH) signaling mutually inhibit each other in cortical RG cells. We provide evidence that ERK signaling is elevated in cortical RG cells during development and evolution. We propose that the expansion of the mammalian cortex, notably in human, is driven by the ERK-BMP7-GLI3R signaling pathway in cortical RG cells, which participates in a positive feedback loop through antagonizing SHH signaling. We also propose that the relatively short cortical neurogenic period in mice is partly due to mouse cortical RG cells receiving higher SHH signaling that antagonizes ERK signaling. Overall design: ? scRNA-Seq of the E14.5 cortex of control mice (Map2k1 and Map2k2 flox), E14.5 cortex of Emx1-Cre; Map2k1/2-dcko mice, E14.5 cortex of hGFAP-Cre; RosaSmoM2 mice. The whole mouse cortex at E14.5 was dissected out under a microscope for scRNA-Seq analysis. Briefly, mouse embryos were dissected out and immediately submerged in fresh ice-cold Hanks balanced salt solution (Gibco 12175-095). The cortex was then cut into 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. The Chromium droplet-based sequencing platform (10X Genomics) was employed to generate scRNA-Seq libraries, following the manufacturer's instructions (manual document part number: CG00052 Rev C). Cellular quality control thresholds were set at 750-5000 genes and <10% mitochondrial transcripts per cell. After filtering, the number of cells in our dataset were as follows: E14.5 control cortex, 8649 cells, 1390 genes/cell; E14.5 Emx1-Cre; Map2k1/2-dcko cortex, 5811 cells, 1713 genes/cell; E14.5 hGFAP-Cre; RosaSmoM2 cortex, 11868 cells, 2663 genes/cell. ? Bulk RNA-Seq data of E14.5 cortex of control mice (n =5), E14.5 cortex of hGFAP-Cre; RosaFgf8 mice (n = 5), E14.5 cortex of hGFAP-Cre; RosaMEK1DD mice (n = 4). The whole cortex from E14.5 control mice (n = 5), from E14.5 hGFAP-Cre; RosaFgf8 mice (n = 5), and from E14.5 hGFAP-Cre; RosaMEK1DD mice (n = 4) was dissected. Total RNA was purified with a mini RNA isolation kit (ZymoGenetics). RNA-seq was performed as recommended by the manufacturer (Illumina). Levels of gene expression were reported in fragments per kilobase of exon per million fragments mapped (FPKM). A gene was considered to be expressed when it had FPKM > 1. For a gene to be called as differentially expressed, it required p < 0.05.