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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.

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. The relationship between regulatory elements, chromatin interactions and gene expression during development remains poorly understood. Here the authors present Tiled-C, a low-input 3C approach to study genome architecture at high resolution, and apply it to mouse erythroid differentiation in vivo, finding that enhancer-promoter interactions are formed gradually during differentiation, concomitant with progressive upregulation of gene activity.

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 . Single-cell transcriptomics, fate assays and a computational theory enable prediction of cell fates during haematopoiesis, discovery of regulators of erythropoiesis and reveal coupling between the erythroid, basophil and mast cell fates. The hierarchy of blood cell lineages Allon Klein, Merav Socolovsky and colleagues examine the emergence of distinct blood cell lineages from mouse haematopoietic progenitors. Their approach combines single-cell transcriptomics, cell fate potential assays and population balance analysis—a computational method for predicting cell fate probabilities from population snapshots. They use a new flow-cytometry strategy to sort cells with newly defined markers of erythroid differentiation and validate the findings at the single-cell level. The results show that differentiation is a continuous, albeit hierarchical, process. They also reveal that erythroid and mast cell fates are coupled, and that remodelling the expression of cell cycle regulators is very important as erythroid cells proceed to terminal differentiation.

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. Maturing erythroblasts become smaller with every cell division. Here, the authors show that Epo stimulation promotes cell division and also generates larger red cells, and that this occurs in mouse and human cells, suggesting that red cell size could be a diagnostic marker for hypoxic stress.

Red blood cell production is regulated by erythropoietin (Epo), maintaining tissue oxygen tension in the steady state and in response to stress. To date, only a handful of factors other than Epo are known to stimulate erythropoiesis, limiting therapeutic options. We recently found that IL-17, a pleiotropic pro-inflammatory cytokine, interacts synergistically with Epo to increase formation of erythroid colonies in vitro. Here, we administered IL-17 to mice to determine whether it accelerates erythropoiesis in vivo. We found that while IL-17 alone had little effect on erythroid and other hematopoietic lineages, combined treatment with both IL-17 and Epo generated a specific and strong synergistic response in erythroid progenitors that significantly increased erythropoietic rate. IL-17 administration also accelerated the erythropoietic response of mice to hypoxia. Single-cell transcriptomic analysis showed that IL-17 acts by sensitizing erythroid progenitors to Epo, rather than through a distinct transcriptional response. Using a dynamical model, we propose that this mechanism optimizes conflicting requirements in the regulation of erythropoiesis, balancing the need for low-cost maintenance of the steady state, with a sufficiently fast stress response. Further, our findings suggest a potentially broadly applicable mechanism whereby pleiotropic cytokines are able to exert lineage-specific effects when their actions are dependent on synergism with lineage-specific factors.

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 β-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.

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the β-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57(KIP2) and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a \"synchromesh\" mechanism that \"locks\" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.

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.

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.

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.