Spatio-temporal adaptation of DOT1L-mediated transcriptional control within defined cell lineages during cortical development

Purpose:Transcriptional regulation of cell fate determination is cortical development. The goals of this study were to determine the role of the histone methyltransferase DOT1L neurogenesis during cortical development in mouse, effect of DOT1L inhibition on lineage progression, and gene regulatory alterations mediated by DOT1L inhibition Methods: Neural progenitor cells in the ventricular zone of E14.5 organotypic brain slices from mouse embryos were labelled via microinjection (Taverna et al., 2012) and treated with DOT1L inhibitor EPZ5676 (Pinometostat) or vehicle for 24 h. Labelled cells and progeny were processed for single cell RNA sequencing (scRNA-seq) using the CEL-Seq2 (Hashimshony, 2016) with modifications as descried in the mCEL-seq2 protocol (Herman et al., 2018). Libraries were sequenced (paired-end) on Illumina Hi-seq 2500 at a depth of ~150,000 reads per cell. Reads were aligned to the mouse transcriptome (GRCm38/mm10) using bwa (version 0.6.2-r126) (H. Li & Durbin, 2009) with default parameters. Cells from two microinjection experiments were pooled for sequencing. Results:710 cells expressing 34,205 genes were recovered. Analysis was performed with Seurat v3.0. The top 800 higjly variable genes were used for clustering recovering 5 cell types: apical progenitors, basal progenitors, transitory cells, neurons I, and neurons II, which were observed in both inhibition and control conditions. Slingshot pseudotime analysis revealed alterations to differentiation trajectories in inhibition condition compared to control. In addition, gene regulatory network analysis revealed alterations in transcription factor activities which were important for metabolic regulation of neurogenesis. Conclusions: This study revealed a novel cell state, transitory cells, which arises after apical progenitor mitoses. It showed that methyltansferase activity of DOT1L is crucial for cortical neurogenesis. Samples from two microinjection experiments were pooled for sequencing. EPZ treated and control cells were pooled to generate one single cell gene expression matrix.