Transcriptome analysis of early stage of neurogenesis reveals regulatory gene network for preplate neuron differentiation and CR cell specification

Background: Preplate is a transient layer critical for cortex development. Cajal-Retzius (CR) cells are the major cell type that later restrict to layer 1 and play important role in organizing cortical layering architecture. However, the molecular mechanisms underlying the generation of preplate and CR cell differentiation remain poorly understood. Results: We used Ebf2-EGFP transgenic mouse that labeled preplate first then persisted to CR, to purify Ebf2 positive cells at different early embryonic stages. Bioinformatic analysis of high throughput RNA-seq revealed differentially expressed genes profile in Ebf2+ and Ebf2- cell population and temporal gene expression dynamics during early neurogenesis and CR neuron specification. Furthermore, our single cell RNA-seq unraveled E15.5 CR cells could be classified into three subpopulations based on their mature states. We also found that the upstream regulatory regions of Ebf2+ cell-enriched genes and lncRNAs were associated with active histone modifications by ChIP-seq studies, indicating epigenetic involvements in early differentiating neuron gene expression. Combined with functional prediction analysis, we identified a number of coding genes and lncRNAs that might regulate different aspects of CR neuron differentiation via primary cell culture, wholemount culture and In Utero Electroporation layer 1 assays. Conclusions: Our work has identified genes and provided histone modification atlas that are related to early neurogenesis, particularly CR neuron identity, and will help understand the molecular mechanisms governing the developmental function of CR neurons. Keywords: Early Neurogenesis, Preplate, Cajal-Retzius neuron development, RNA-seq, Single-cell RNA-seq, Histone modification, ChIP-seq, LncRNAs