Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation. Author summary Neural stem cells and progenitor cells in the embryonic brain give rise to neurons following a precise temporal order after initial expansion. Early-born neurons including Cajal-Retzius (CR) cells and subplate neurons form the preplate in the developing cerebral cortex, then CR neurons occupy the layer 1, playing an important role in cortical histogenesis. The molecular mechanisms governing the early neuronal differentiation processes remain to be explored. Here, by genome-wide approaches including bulk RNA-seq, single-cell RNA-seq and ChIP-seq, we comprehensively characterized the temporal dynamic gene expression profile and epigenetic status at different stages during early cortical development and uncovered molecularly heterogeneous subpopulations within the CR cells. We revealed CR neuron signatures and cell type-specific histone modification patterns along early neuron specification. Using in vitro and in vivo assays, we identified novel lncRNAs as potential functional regulators in preplate differentiat