• Transcriptome analyses of human and murine reveal significant stage and speciesspecific differences across stages of terminal erythroid differentiation.• These transcriptomes provide a significant resource for understanding mechanisms of normal and perturbed erythropoiesis.We recently developed fluorescence-activated cell sorting (FACS)-based methods to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. We used these techniques to obtain pure populations of both human and murine erythroblasts at distinct developmental stages. RNA was prepared from these cells and subjected to RNA sequencing analyses, creating unbiased, stage-specific transcriptomes. Tight clustering of transcriptomes from differing stages, even between biologically different replicates, validated the utility of the FACSbased assays. Bioinformatic analyses revealed that there were marked differences between differentiation stages, with both shared and dissimilar gene expression profiles defining each stage within transcriptional space. There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes. Clustering and network analyses revealed that varying stage-specific patterns of expression observed across differentiation were enriched for genes of differing function. Numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development in various inherited and acquired erythroid disorders. (Blood. 2014;123(22):3466-3477)
IntroductionMammalian erythropoiesis is an excellent example of the complex changes in temporal, developmental, and differentiation stage-specific gene expression exhibited by a single cell type.1,2 In the mammalian embryo and fetus, erythroid cells have differing developmental origins, with the primitive erythroid cell lineage developing from yolk sac-derived erythroid progenitors, and the definitive cell lineage maturing from 2 different developmentally regulated stem and progenitor cell populations. [3][4][5][6] These cells have different programs of regulation, with variation in spatial, temporal, and site-specific differentiation.In the adult, mature erythrocytes are the terminally differentiated final cellular product derived from hematopoietic stem and progenitor cells (HSPC). HSPCs undergo a series of lineage choice fate decisions, with increasingly restricted potential, ultimately committing to the erythroid lineage and beginning erythropoiesis. Traditionally, erythropoiesis has been divided into 3 stages: early erythropoiesis, terminal erythroid differentiation, and reticulocyte maturation.2 Early erythropoiesis involves commitment of multi-lineage progenitors into erythroid progenitor cells, with proliferation and d...