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• 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...

Two-component signaling systems, involving His kinases, His-containing phosphotransfer proteins, and response regulators, have been implicated in plant responses to hormones and environmental factors. Genomic analysis of Arabidopsis supports the existence of 22 response regulators (ARRs) that can be divided into at least two distinct groups designated type-A and type-B. Phylogenetic analysis indicates that the type-B family is composed of one major and two minor subfamilies. The expression of the type-B ARRs was examined by using both reverse transcription-PCR and b-glucuronidase fusion constructs. The major subfamily of type-B ARRs showed particularly high expression in regions where cytokinins play a significant role, including cells in the apical meristem region and in young leaves that would be undergoing cell division. Multiple members within this same subfamily of type-B ARRs were expressed near the root tip with highest expression in the root elongation zone. b-Glucuronidase-fusions to full-length ARR2, ARR12, and ARR19 were nuclear localized, consistent with a role in transcriptional regulation. These data suggest that differing expression levels of the type-B ARRs may play a role in modulating the cellular responses to cytokinin.Plant two-component signaling systems have been implicated in vital cellular processes such as the responses to cytokinins, ethylene, red light, and osmosensing (Schaller, 2000;Hutchison and Kieber, 2002;Hwang et al., 2002;Schaller et al., 2002). Twocomponent systems were originally identified in bacteria, and in their simplest form involve a receptor kinase that autophosphorylates itself on a conserved His residue in response to an environmental stimulus (Mizuno, 1997;Stock et al., 2000). This phosphate is then transferred to a conserved Asp residue within the receiver domain of a response regulator. Phosphorylation of the response regulator modulates its ability to mediate downstream signaling in the pathway. Of particular relevance to plants is a permutation on the twocomponent system known as the multi-step phosphorelay (Swanson et al., 1994;Schaller, 2000). The multi-step phosphorelay makes use of three components: a hybrid receptor kinase that contains both His-kinase and receiver domains in one protein, a His-containing phosphotransfer (HPt) protein, and a separate response regulator. In these multi-step phosphorelays the phosphate is transferred from amino acid to amino acid in sequence His to Asp to His to Asp.In Arabidopsis, proteins with homology to all elements of the two-component system have been identified, including His kinases, response regulators, and HPt proteins (Schaller, 2000(Schaller, , 2002Hutchison and Kieber, 2002;Hwang et al., 2002). Phosphorylation activity has been confirmed in each case. Thus, all elements needed to establish a histidyl-aspartyl phosphorelay are represented in plants. Analysis of the Arabidopsis genome reveals the existence of 8 His kinases, 22 response regulators, and 5 HPt proteins that contain all the conserved residues required ...