Transcriptomic profiling of subpopulations of mouse embryonic subplate neurons.

Subplate neurons (SpNs) are among the earliest-born and maturing neurons in the developing cerebral cortex. They arise from multiple origins and can be classified into several subgroups based on morphology, connectivity, and gene expression. These neurons play essential roles in cortical circuit formation, yet their cellular diversity and transcriptional dynamics remain incompletely understood. Here, we characterized transcriptomic profiles of SpN subpopulations in embryonic mouse cortex using Lpar1-EGFP and NeuroD1/Cre-ERT2 (D1B) reporter lines. We applied complementary approaches of gene expression profiling, including bulk microarray analysis, single-cell RNA sequencing (scRNA-seq), and Visium spatial transcriptomics. At embryonic day 17 (E17), scRNA-seq identified 10 distinct Lpar1-EGFP-positive SpN clusters, which spatial transcriptomics mapped to specific cortical regions. While many markers showed enrichment within the subplate region, others extended into the hippocampus and ventral pallium (including the amygdala, claustrum, and endopiriform nucleus). Integrated analysis of Lpar1-EGFP and D1B lines revealed both overlapping and unique gene expression signatures, highlighting dynamic markers of subplate identity. Comparisons between E15 and E17 datasets showed substantial transcriptional shifts, suggesting rapid developmental changes in SpN subgroups. Validation with in situ hybridization and RNAscope confirmed the selectivity of key markers, including Cryab, Cdh13, Nr4a2, and Lmo3. Together, these findings provide a molecular framework for further classifying SpN subtypes and identifying candidate markers for transient versus persistent populations, thereby advancing our understanding of early cortical development.