Integrating Single-Cell Transcriptomics with Phosphoproteomics Dissects the EGFR-Dependent Network Governing Cortical Gliogenesis
Deciphering gliogenesis is fundamental to understanding glial biology. Epidermal growth factor receptor (EGFR) plays an essential role in this process, with its expression initiating at the onset of gliogenesis and peaking perinatally, coinciding with key stages of glial development. Conditional ablation of EGFR in the glial lineage resulted in a pronounced reduction of cortical glia, particularly astrocytes. To systematically map the EGFR-dependent molecular landscape during gliogenesis, we generated an integrated multi-omics atlas through lineage-restricted deletion of Egfr using hGFAP-Cre. This high-resolution resource delineates the transcriptional and phosphoproteomic reprogramming of radial glial lineages upon EGFR loss. We profiled 24,738 cells at embryonic day 18 (E18) and 18,343 cells at postnatal day 2 (P2) from control and Egfr-CKO cortices by single-cell RNA sequencing. In parallel, phosphoproteomic analysis quantified 6,409 high-confidence phosphorylation sites, 6,183 unique phosphopeptides, and 2,510 phosphorylated proteins. Together, these datasets establish a comprehensive framework for dissecting EGFR-dependent regulatory networks and signaling crosstalk, providing mechanistic insight into glial fate specification and potential developmental origins of gliopathies. Overall design: Egfr-CKO embryos were subjected to lateral ventricle injection of Cell Trace Yellow at embryonic day 16 (E16) . The cerebral cortices were harvested at E18, corresponding to the injection timepoints. Fluorescent dye-labeled cells were isolated via fluorescence-activated cell sorting using a BD FACSAriaII flow cytometer.