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

PLoS Genet. 2021 Mar 24;17(3):e1009355. doi: 10.1371/journal.pgen.1009355. eCollection 2021 Mar.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Biomarkers
  • Cell Differentiation* / genetics
  • Cells, Cultured
  • Cerebral Cortex / metabolism
  • Gene Expression
  • Gene Expression Regulation
  • Genes, Reporter
  • Genetic Heterogeneity
  • Genomics* / methods
  • Histones
  • Immunohistochemistry
  • Mice
  • Mice, Transgenic
  • Neurogenesis / genetics*
  • Neurons / cytology
  • Neurons / metabolism*
  • RNA, Long Noncoding / genetics
  • Single-Cell Analysis
  • Temporal Lobe / metabolism*
  • Transcription Factors
  • Transcription Initiation Site


  • Basic Helix-Loop-Helix Transcription Factors
  • Biomarkers
  • Ebf2 protein, mouse
  • Histones
  • RNA, Long Noncoding
  • Transcription Factors

Grant support

This work was supported by the Ministry of Science and Technology (2019YFA0110102 to Q.S.), the National Natural Science Foundation of China (31170247 to Q.S.) the Fundamental Research Funds for the Central Universities (22120200411 to Q.S.), the National Key R&D Program of China (2018YFA0108600 to Q.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.