Pas de deux: the coordinated coupling of erythroid differentiation with the cell cycle.

PURPOSE OF REVIEW: Recent work reveals that cell cycle duration and structure are remodeled in lock-step with distinct stages of erythroid differentiation. These cell cycle features have regulatory roles in differentiation, beyond the generic function of increasing cell number. RECENT FINDINGS: Developmental progression through the early erythroid progenitor stage (known as colony-forming-erythroid, or 'CFU-e') is characterized by gradual shortening of G1 phase of the cycle. This process culminates in a key transcriptional switch to erythroid terminal differentiation (ETD) that is synchronized with, and dependent on, S phase progression. Further, the CFU-e/ETD switch takes place during an unusually short S phase, part of an exceptionally short cell cycle that is characterized by globally fast replication fork speeds. Cell cycle and S phase speed can alter developmental events during erythroid differentiation, through pathways that are targeted by glucocorticoid and erythropoietin signaling during the erythroid stress response. SUMMARY: There is close inter-dependence between cell cycle structure and duration, S phase and replication fork speeds, and erythroid differentiation stage. Further, modulation of cell cycle structure and speed cycle impacts developmental progression and cell fate decisions during erythroid differentiation. These pathways may offer novel mechanistic insights and potential therapeutic targets.

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

The role of specialized cell cycles during erythroid lineage development: insights from single-cell RNA sequencing.

Early erythroid progenitors known as CFU-e undergo multiple self-renewal cell cycles. The CFU-e developmental stage ends with the onset of erythroid terminal differentiation (ETD). The transition from CFU-e to ETD is a critical cell fate decision that determines erythropoietic rate. Here we review recent insights into the regulation of this transition, garnered from flow cytometric and single-cell RNA sequencing studies. We find that the CFU-e/ETD transition is a rapid S phase-dependent transcriptional switch. It takes place during an S phase that is much shorter than in preceding or subsequent cycles, as a result of globally faster replication forks. Furthermore, it is preceded by cycles in which G1 becomes gradually shorter. These dramatic cell cycle and S phase remodeling events are directly linked to regulation of the CFU-e/ETD switch. Moreover, regulators of erythropoietic rate exert their effects by modulating cell cycle duration and S phase speed. Glucocorticoids increase erythropoietic rate by inducing the CDK inhibitor p57KIP2, which slows replication forks, inhibiting the CFU-e/ETD switch. Conversely, erythropoietin promotes induction of ETD by shortening the cycle. S phase shortening was reported during cell fate decisions in non-erythroid lineages, suggesting a fundamentally new developmental role for cell cycle speed.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

The shifting shape and functional specializations of the cell cycle during lineage development.

Essentially all cell cycling in multicellular organisms in vivo takes place in the context of lineage differentiation. This notwithstanding, the regulation of the cell cycle is often assumed to be generic, independent of tissue or developmental stage. Here we review developmental-stage-specific cell cycle adaptations that may influence developmental decisions, in mammalian erythropoiesis and in other lineages. The length of the cell cycle influences the balance between self-renewal and differentiation in multiple tissues, and may determine lineage fate. Shorter cycles contribute to the efficiency of reprogramming somatic cells into induced pluripotency stem cells and help maintain the pluripotent state. While the plasticity of G1 length is well established, the speed of S phase is emerging as a novel regulated parameter that may influence cell fate transitions in the erythroid lineage, in neural tissue and in embryonic stem cells. A slow S phase may stabilize the self-renewal state, whereas S phase shortening may favor a cell fate change. In the erythroid lineage, functional approaches and single-cell RNA-sequencing show that a key transcriptional switch, at the transition from self-renewal to differentiation, is synchronized with and dependent on S phase. This specific S phase is shorter, as a result of a genome-wide increase in the speed of replication forks. Furthermore, there is progressive shortening in G1 in the period preceding this switch. Together these studies suggest an integrated regulatory landscape of the cycle and differentiation programs, where cell cycle adaptations are controlled by, and in turn feed back on, the propagation of developmental trajectories. This article is categorized under: Congenital Diseases > Stem Cells and Development.

Dynamics of the 4D genome during in vivo lineage specification and differentiation.

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.

Role of Interferon-γ-Producing Th1 Cells in a Murine Model of Type I Interferon-Independent Autoinflammation Resulting From DNase II Deficiency.

OBJECTIVE: Patients with hypomorphic mutations in DNase II develop a severe and debilitating autoinflammatory disease. This study was undertaken to compare the disease parameters in these patients to those in a murine model of DNase II deficiency, and to evaluate the role of specific nucleic acid sensors and identify the cell types responsible for driving the autoinflammatory response. METHODS: To avoid embryonic death, Dnase2-/- mice were intercrossed with mice that lacked the type I interferon (IFN) receptor (Ifnar-/- ). The hematologic changes and immune status of these mice were evaluated using complete blood cell counts, flow cytometry, serum cytokine enzyme-linked immunosorbent assays, and liver histology. Effector cell activity was determined by transferring T cells from Dnase2-/- × Ifnar-/- double-knockout (DKO) mice into Rag1-/- mice, and 4 weeks after cell transfer, induced changes were assessed in the recipient mice. RESULTS: In Dnase2-/- × Ifnar-/- DKO mice, many of the disease features found in DNase II-deficient patients were recapitulated, including cytopenia, extramedullary hematopoiesis, and liver fibrosis. Dnase2+/+ × Rag1-/- mice (n > 22) developed a hematologic disorder that was attributed to the transfer of an unusual IFNγ-producing T cell subset from the spleens of donor Dnase2-/- × Ifnar-/- DKO mice. Autoinflammation in this murine model did not depend on the stimulator of IFN genes (STING) pathway but was highly dependent on the chaperone protein Unc93B1. CONCLUSION: Dnase2-/- × Ifnar-/- DKO mice may be a valid model for exploring the innate and adaptive immune mechanisms responsible for the autoinflammation similar to that seen in DNASE2-hypomorphic patients. In this murine model, IFNγ is required for T cell activation and the development of clinical manifestations. The role of IFNγ in DNASE2-deficient patient populations remains to be determined, but the ability of Dnase2-/- mouse T cells to transfer disease to Rag1-/- mice suggests that T cells may be a relevant therapeutic target in patients with IFN-related systemic autoinflammatory diseases.

From blood development to disease: a paradigm for clinical translation.

Translating basic research to the clinic is a primary aim of Disease Models & Mechanisms, and the recent successes in hematopoiesis research provide a blueprint of how fundamental biological research can provide solutions to important clinical problems. These advances were the main motivation for choosing hematopoiesis disorders as the focus of our inaugural meeting, 'Blood Disorders: Models, Mechanisms and Therapies', which was held in early October 2019. This Editorial discusses the reasons for and the challenges of interdisciplinary research in hematopoiesis, provides examples of how research in model systems is a key translational step towards effective treatments for blood disorders and summarizes what the community believes are the key exciting developments and challenges in this field.

Population snapshots predict early haematopoietic and erythroid hierarchies.

The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.

High-throughput single-cell fate potential assay of murine hematopoietic progenitors in vitro.

The advent of single-cell transcriptomics has led to the proposal of a number of novel high-resolution models for the hematopoietic system. Testing the predictions generated by such models requires cell fate potential assays of matching, single-cell resolution. Here we detail the development of an in vitro high-throughput single-cell culture assay using flow cytometrically sorted single murine bone marrow progenitors, which measures their differentiation into any of five myeloid lineages. We identify critical parameters for single-cell culture outcome, including the choice of sorter nozzle size and pressure, culture media, and the coating of culture dishes with extracellular matrix proteins. Further, we find that accurate assay readout requires the titration of antibodies specifically for their use under low-cell-number conditions. Our approach may be used as a template for the development of single-cell fate potential assays for a variety of blood cell progenitors.

Fundamental limits on dynamic inference from single-cell snapshots.

Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements.

Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch.

Cell cycle regulators are increasingly implicated in cell fate decisions, such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. We studied an S phase-dependent cell fate switch, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. We found that progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, a novel function for cyclin-dependent kinase inhibitors. The switch to differentiation entails rapid down-regulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. Our work suggests that cell cycles with specialized global DNA replication dynamics are integral to the maintenance of specific cell states and to cell fate decisions.

Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells.

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

Erythropoiesis: from molecular pathways to system properties.

Erythropoiesis is regulated through a long-range negative feedback loop, whereby tissue hypoxia stimulates erythropoietin (Epo) secretion, which promotes an increase in erythropoietic rate. However, this long-range feedback loop, by itself, cannot account for the observed system properties of erythropoiesis, namely, a wide dynamic range, stability in the face of random perturbations, and a rapid stress response. Here, we show that three Epo-regulated erythroblast survival pathways each give rise to distinct system properties. The induction of Bcl-xL by signal transducer and activator of transcription 5 (Stat5) is responsive to the rate of change in Epo levels, rather than to its absolute level, and is therefore maximally but transiently activated in acute stress. By contrast, Epo-mediated suppression of the pro-survival Fas and Bim pathways is proportional to the levels of stress/Epo and persists throughout chronic stress. Together, these elements operate in a manner reminiscent of a "proportional-integral-derivative (PID)" feedback controller frequently found in engineering applications. A short-range negative autoregulatory loop within the early erythroblast compartment, operated by Fas/FasL, filters out random noise and controls a reserve pool of early erythroblasts that is poised to accelerate the response to acute stress. Both these properties have previously been identified as inherent to negative regulatory motifs. Finally, we show that signal transduction by Stat5 combines binary and graded modalities, thereby increasing signaling fidelity over the wide dynamic range of Epo found in health and disease.

Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and graded modalities combine to generate high-fidelity Stat5 signaling over the entire basal and stress Epo range. They suggest that dynamic behavior may encode information during STAT signal transduction.

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways.

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and ß-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway "resets," allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using "knock-in" mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a "stop-gap" in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.

Global DNA demethylation during mouse erythropoiesis in vivo.

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific ß-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.