GSE167576
Coordinated Changes in Gene Expression Kinetics Underlie both Mouse and Human Erythroid Maturation
Background: Single cell molecular profiling technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single cell RNA-Seq permits prediction of future expression states. Here we applied this 'so-called RNA velocity concept' to an extended timecourse dataset covering mouse gastrulation and early organembryogenesis. Results: Intriguingly, RNA velocity correctly identified epiblast cells as the starting point, but several This analysis revealed major limitations of the current RNA velocity framework, whereby application to extended timecourse data resulted in trajectory predictions at later stages were inconsistentmpatible with both real time ordering and existing knowledge. The most striking discrepancy concerned red blood cell maturation, with velocity-inferred trajectories diametrically opposinged to the true differentiation path. Investigating the underlying causes revealed a group of genes with a coordinated step-changeboost in transcriptionexpression kinetics, thus violating the assumptions behindframework of current velocity analysis suites, which does not accommodate time-dependent kinetics changes in expression dynamicsover a lineage. Using scRNA-Seq analysis of chimaeric mouse embryos lacking the major erythroid master regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be up-regulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. e expected block in erythroid maturation as well as induction of PU.1 and expansion of megakaryocyte progenitors. MoreoverIn addition to, the expected block in erythroid maturation, the Gata1- chimera dataset revealed induction of PU.1 and expansion of megakaryocyte progenitors. genes with complex expression kinetics in wild type cells fail to progress to the late erythroid boost in kinetics, thus underscoring the coordination in modulating expression kinetics. Finally, we show that erythropoiesiserythroid maturation  in human fetal liver is similarly characterized by a coordinated step-changes in gene expression kinetics. Conclusions: By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal that erythroid maturation is characterized by a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes. Overall design: single-cell RNA sequencing of Gata1- embryonic chimeras