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

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 heterogeneity 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. mRNA profile of cortical Ebf2-EGFP+ and Ebf2-EGFP- cells at mouse embryonic stage E11.5, E13.5 and E15.5发育中新皮层的神经发生起始于前板（preplate）的形成，前板由早期诞生的神经元组成，包括Cajal-Retzius（CR）细胞与皮层下板神经元。本研究利用Ebf2-EGFP转基因小鼠——该小鼠的增强绿色荧光蛋白（EGFP）最初标记前板神经元，后续持续表达于CR细胞中——揭示了早期神经发生与CR细胞分化的动态转录组图谱。通过对多个早期神经发生阶段开展全基因组RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq）分析，本研究阐明了早期神经发生的时序基因表达动态，以及早期分化神经元中独特的组蛋白修饰模式。本研究鉴定出一批参与早期神经元分化的编码基因与长链非编码RNA（lncRNAs），并通过体外（in vitro）与体内（in vivo）功能实验对其进行了验证。此外，在小鼠胚胎发育第15.5天（E15.5），此时Ebf2-EGFP阳性细胞大多为CR神经元，对纯化的Ebf2-EGFP阳性细胞进行单细胞测序分析，揭示了CR神经元的分子异质性，但未发现具有不同区域起源的细胞形成明显聚类。通过伪时间轨迹分析，这些细胞可被分为三种不同的发育状态，揭示了在CR神经元身份与功能建立过程中，从早期通用神经元分化到晚期命运特化的遗传级联反应。本研究结果为阐明皮层发育过程中早期分化步骤（尤其是CR神经元分化）的分子机制提供了新的见解。本数据集包含小鼠胚胎发育阶段E11.5、E13.5及E15.5时皮层Ebf2-EGFP阳性与阴性细胞的mRNA表达谱。