Single cell 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 ‘RNA velocity concept’ to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Intriguingly, RNA velocity correctly identified epiblast cells as the starting point, but several trajectory predictions at later stages were inconsistent with both real time ordering and existing knowledge. The most striking discrepancy concerned red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes revealed a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid 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. In addition to the expected block in erythroid maturation, the Gata1- chimera dataset revealed induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

Background Single-cell 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 apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Results Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. Conclusions By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

BackgroundThe fetal hypothalamic-pituitary-adrenal (HPA) axis plays a key role in the control of parturition and maturation of organ systems in preparation for birth. In hypothyroid fetuses, gestational length may be prolonged and maturational processes delayed. The extent to which the effects of thyroid hormone deficiency in utero on the timing of fetal maturation and parturition are mediated by changes to the structure and function of the fetal HPA axis is unknown. MethodsIn twin sheep pregnancies where one fetus was thyroidectomized and the other shamoperated, this study investigated the effect of hypothyroidism on circulating concentrations of adrenocorticotrophic hormone (ACTH) and cortisol, and the structure and secretory capacity of the anterior pituitary and adrenal glands. The relative population of pituitary corticotrophs, and the masses of the adrenal zones, were assessed by immunohistochemical and stereological techniques. Adrenal mRNA abundances of key steroidogenic enzymes and growth factors were examined by qPCR. ResultsHypothyroidism in utero reduced plasma concentrations of ACTH and cortisol. In thyroiddeficient fetuses, the mass of corticotrophs in the anterior pituitary gland was unexpectedly increased, while the mass of the zona fasciculata and its proportion of the adrenal gland were decreased. These structural changes were associated with lower adrenocortical mRNA abundances of insulin-like growth factor-I (IGFI) and its receptor, and key steroidogenic 3 enzymes responsible for glucocorticoid synthesis. The relative mass of the adrenal medulla and its proportion of the adrenal gland were increased by thyroid hormone deficiency in utero, without any change in expression of phenylethanolamine N-methyltransferase or the IGF system. ConclusionsThyroid hormones are important regulators of the structure and secretory capacity of the pituitary-adrenal axis before birth. In hypothyroid fetuses, low plasma cortisol may be due to impaired adrenocortical growth and steroidogenic enzyme expression, secondary to low circulating ACTH concentration. Greater corticotroph population in the anterior pituitary gland of the hypothyroid fetus indicates compensatory cell proliferation and that there may be abnormal corticotroph capacity for ACTH synthesis and/or impaired hypothalamic input. Suppression of the development of the fetal HPA axis by thyroid hormone deficiency may contribute to the delay in fetal maturation and delivery observed in hypothyroid offspring.