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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.

The manner by which multipotent hematopoietic progenitors commit to the erythroid lineage, and the subsequent processes that govern early erythroid progenitor development, are not well understood. Part of the challenge for investigating these was the lack of a rigorous strategy for isolating directly from tissue the early erythroid progenitors, which are functionally defined as the cell 'units' that give rise to erythroid colonies (CFU-e) or bursts (BFU-e) in culture. Indeed, the early erythroid trajectory, that starts with multi-potential progenitors and gives rise to BFU-e, CFU-e and to erythroblasts undergoing terminal differentiation, was not fully elucidated. We addressed these gaps using single cell transcriptomics, combined with functional assays that validated computational predictions 1. These showed that early hematopoietic progenitors form a continuous, hierarchical branching structure, in which the erythroid and basophil/mast cell fates are unexpectedly coupled. We delineated a novel flow-cytometric strategy that prospectively isolates CFU-e and BFU-e progenitors with high purity, and in combination with computational predictions, identified novel growth factor receptors that regulate early erythropoiesis. We further discovered that early erythroid development entails profound remodeling of both G1 and S phases of the cycle, resulting in cell cycle specializations that orchestrate the developmental process, including a gradual shortening of G1 during the CFU-e phase, followed by a sharp increase in the speed of S phase during the S-phase dependent activation of the erythroid terminal differentiation program 1-3(Figure 2). 1. Tusi BK, Wolock SL, Weinreb C, et al. Population snapshots predict early haematopoietic and erythroid hierarchies. Nature. 2018;555(7694):54-60. 2. Hwang Y, Futran M, Hidalgo D, et al. Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch. Science Advances. 2017;3:e1700298. 3. Pop R, Shearstone JR, Shen Q, et al. A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol. 2010;8(9). Disclosures No relevant conflicts of interest to declare.

Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR−/−progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR−/− progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR−/− and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.© 1998 by The American Society of Hematology.

Erythroid differentiation may be divided into two broad stages: early development, and terminal differentiation. Early development was first explored using the colony-formation potential of hematopoietic tissue. This approach identified multi-potential progenitors (MPP) and unipotential erythroid progenitors that form 'bursts' (BFUe) and smaller colonies (CFUe). Early erythroid development is followed by erythroid terminal differentiation (ETD), which profoundly remodels erythroblasts into enucleated red cells. The molecular study of ETD was fundamentally transformed with the development of cell-surface marker strategies that identify sequential stage-specific erythroblasts in hematopoietic tissue. By contrast, there have been no strategies that systematically identify the entire cellular and molecular trajectory of the early erythroid lineage as it first arises from the MPP and progresses to the point where the ETD program is activated. To address this gap, we undertook single-cell transcriptomics using the InDrop-seq platform (Klein et al. Cell 161:1187 2015). We analyzed Kit+ cells in the bone marrow of mice in the basal state, mice stimulated with Erythropoietin (Epo) for 48 hours, or fetal liver cells. We used Next-Generation Sequencing data to construct k-nearest neighbor (kNN) graphs of cell states for each condition, and extracted the erythroid trajectory using Population Balance Analysis (PBA), a novel computational approach that predicts differentiation fates from single cell RNA profiles. We identified early erythroid developmental stages based on the modeled probability of erythroid commitment, gene expression dynamics, and the architecture of the kNN graph. By screening for appropriate cell surface markers, we developed a flow-cytometric strategy that isolates sub-populations corresponding to regions of the kNN graph, including sub-regions of the erythroid trajectory. This allowed validation of gene expression patterns and of cell fate predictions using in vitro colony formation assays. The earliest stage in the erythroid trajectory, the Erythroid/Basophil MPP stage (E/B-MPP) contains progenitors that emerge from MPPs, predicted to remain mutli-potential but be biased towards bipotential erythroid/basophil and eryrthroid/megakaryocytic fates. This stage is characterized by rapidly changing gene expression profiles. Genes whose expression correlates with the probability of erythroid commitment include both known transcriptional regulators of erythropoiesis such as GATA1, GATA2, Ldb1 and Klf1, as well as novel candidates. Downstream from the E/B-MPP, the two-dimensional projection of the kNN graph becomes a narrow bottleneck, indicating a transient stage. Here the erythroid trajectory contains cells with rapidly increasing probability of erythroid commitment (Emerging Erythroid Progenitors, EEP). The bottleneck connects to a bulge-like region in which progenitors have an extremely high probability of attaining the erythroid fate (committed erythroid progenitors, CEP). This region contains the majority of the marrow's committed erythroid progenitors, and functions as an amplification module, increasing in size in Epo-stimulated marrow and in the fetal liver. Functionally, cells in the bottleneck region give rise to multifocal erythroid colonies (early and late BFUe), whereas cells in the CEP amplification module give rise to unifocal erythroid colonies, including the CFUe. Therefore, the ability of a progenitor to give rise to either multifocal or unifocal colonies correlates closely with molecular stage. Cells in the CEP module express a unique set of genes, induced at the module entry, and repressed at its exit. These include growth-factor receptors mst1r, ryk and il17ra. Their ligands, MSP, Wnt5a and IL17a, are novel regulators of erythropoiesis, either stimulating or inhibiting the formation of erythroid colonies. Exit from the CEP module is marked by a rapid switch, in which the repression of CEP genes coincides with induction of the ETD program. Remarkably, this switch is synchronized with expression of G1/S and S phase genes, underlying a role for S phase progression in ETD activation (Pop et al., PLoS Biology 2010). Our work charts the erythroid trajectory of murine hematopoietic tissue, identifying developmental milestones, setting the stage for their molecular study and for discovery of novel erythroid regulators. Disclosures Klein: OneCell Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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. Here we studied an S phase- dependent cell fate switch in the erythroid fetal liver, 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. In the fetal liver, this transition corresponds to the transition from subset S0 (CD71-low, Ter119-negative) to subset S1 (CD71-high, Ter119-negative). We found that the S0 to S1 transition takes place during an S phase that is abruptly shorter (decreasing from 7 hours to 4 hours). Further, self-renewing S0 cells uniquely express the cyclin-dependent kinase (CDK) inhibitor p57KIP2 during S phase. To investigate its potential role, we studied DNA replication in vitro and in vivo in p57KIP2 -deficient fetal liver progenitors, employing a variety of techniques, including DNA combing. We found that S0 erythroid progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, either in vivo, or in dexamethasone-dependent expansion cultures in vitro. The switch from self-renewal in S0 to differentiation in wild-type S1 progenitors entails rapid downregulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. In the absence of p57KIP2, replication fork processivity increases prematurely in self-renewing S0 cells, prior to the activation of the erythroid transcriptional program (Figure 1), resulting in replicative stress and cell death. It is well established that differentiation leads to reprogramming of DNA replication, reflected by changes to origin usage and to the timing of replication of chromatin domains. Here we find that the replication program is fundamentally altered in additional key respects: the global processivity of replication forks, regulated by CDK activity, increases abruptly with the switch from self-renewal to differentiation, affecting DNA synthesis rates and S phase duration. Our results are also of interest since the regulation of replication kinetics was thought to be primarily via the regulation of origin firing efficiency, rather than via fork processivity. Here we found no difference in the former (there was no significant change in inter-origin distances, Figure 1). While the full significance of faster forks to the activation of the erythroid transcriptional program is yet to be understood, a recent report found that T cell help leads to faster forks and a shorter S phase in B cells (Gitlin et al., Science 349, 643-646 2015). Regulation of global fork speed may therefore be an intrinsic part of physiological developmental programs. Disclosures No relevant conflicts of interest to declare.

Fate commitment of hematopoietic progenitor cells (HPCs) is thought to occur through a series of hierarchical fate choices, established over the past four decades through live cell tracking, in vitro colony-forming assays and transplantation of defined sub-populations of HPCs. Depictions of the HPC hierarchy invoke a tree structure, with gradual lineage-restriction at branch points, although the precise tree remains controversial. A second controversy relates to the nature of undifferentiated MPPs. MPPs have been suggested to express conflicting lineage-restricted programs indicating multi-lineage priming, or to host distinct cell sub-populations with intrinsic biases, or alternatively to form an entirely na•ve state. We asked whether the molecular state of cells seen by RNA profiling of thousands of single HPCs could resolve these controversies, by correctly predicting the known fates of sub-populations of HPCs reported over the past two decades, defining MPP heterogeneity, and defining the topology of HPC fate commitment. Predicting the future behavior of cells from high-dimensional snapshots of their current state is an unsolved problem. Until now, snapshots of single cell molecular states have been used to order events in cell differentiation, cell cycle, and perturbation response by methods that fit cell states to a curve or a tree, but these approaches tend to be suggestive rather than predictive. Here, we invoke a conservation law Ð commonly known as "population balance" Ð to formally predict differentiation fates from single cell molecular profiles, using an approach that is asymptotically exact given certain assumptions about the differentiation process. Application of the conservation law to single cell RNA profiles required developing novel mathematical results, leading to an algorithm termed Population Balance Analysis (PBA). We apply PBA to bone marrow hematopoiesis, and recover the structure of the hematopoietic progenitor cell (HPC) population as it differentiates into seven lineages. The inferred fate choices reconcile fate-potential assays from the past two decades, and suggest a unified molecular definition of the hematopoietic hierarchy. In contrast to the canonical hierarchy, we predict that HPCs do not form a strict tree, and that MPPs show evidence of multi-lineage priming with simultaneous low-level gene expression of conflicting differentiation programs. In normal hematopoiesis, we predict a novel erythroid-basophil progenitor cell state, whereas erythroid/megakaryocyte fates become coupled in stress. We predict several novel regulators of cell fate at choice points in hematopoiesis. Overall, this work reconciles novel RNA-Seqdata with gold standard functional fate assays; it predicts novel regulators of hematopoiesis; and it establishes a predictive analytical method for dissecting complex differentiation hierarchies from single cell molecular profiles. Figure Figure. Disclosures Klein: OneCell Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

We previously characterized an S phase-dependent commitment switch in mouse fetal liver. Specifically, the upregulation of CD71 in the transition from Subset 0 (S0, Lin-CD71medium) to Subset 1 (S1, Lin-CD71high) identifies cells in early S phase of the last colony-forming-unit-erythroid (CFUe) generation, undergoing key differentiation events, including onset of Erythropoietin (Epo) dependence, GATA-1 activation and reconfiguration of chromatin at the §-globin locus control region. This commitment switch requires S phase progression, and is regulated by the S-phase- dependent downregulation of the transcription factor PU.1, a GATA-1 antagonist. Of note, the specific cell cycle S phase in which this commitment switch takes place differs from S phase in previous cycles, in that it is shorter, with a 50% faster rate of nucleotide incorporation into DNA. Here, we investigate whether a similar commitment switch takes place in adult bone-marrow. Using fresh bone-marrow from mice in the basal state or following Epo injection, we determined colony-forming potential, transcriptional profiles and cell cycle status of Kit+Lin- subsets defined by cell surface markers that have been previously implicated as enriched in the megakaryocytic-erythrocytic lineages: CD105, CD150, CD55, CD71, in conjunction with a PU.1-GFP reporter mouse. Using quantitative RT-PCR together with flow-cytometric analysis and the SPADE algorithm we identified a linear erythroid developmental sequence of Epo-responsive Kit+Lin-CD55+ bone-marrow subsets, which gradually declined in PU.1, transiently increased CD150, and upregulated both CD105 and CD71. The loss of PU.1 coincided with increased GATA1 levels and the transcriptional activation of erythroid genes. CFUe activity peaked within Kit+Lin-CD55+ cells that were also CD105+, where colonies of other lineage potential were rare (Fig 1). Upregulation of CD71 in erythroid cells indicates the onset of Epo receptor signaling and Epo dependence. We found that in Kit+Lin-CD55+ bone-marrow in vivo, high levels of CD71 were highly correlated with S phase (Fig 2). Furthermore, the rate of incorporation of the nucleotide analogue BrdU was substantially higher in CD71 -high cells, suggesting a rapid, short S phase. To functionally examine the role of S phase, we isolated Kit+Lin-CD55+CD105+ cells that were CD71 low, and followed their differentiation in vitro. These cells upregulate CD71 within 10 to 16 hours, followed by upregulation of Ter119. Upregulation of CD71 was Epo dependent, and took place in S phase; further, it was reversibly inhibited by the DNA polymerase inhibitor, Aphidicolin, suggesting dependence on S phase progression. We conclude that adult bone marrow Kit+Lin-CD55+CD105+ are at the CFUe developmental stage, and undergo an Epo and S-phase dependent commitment switch that activates the erythroid transcriptional and differentiation program. S phase during this commitment switch is characterized by a fast DNA synthesis rate. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

We characterized a rapid commitment switch in mouse fetal liver cells in vivo, that activates the GATA-1 –dependent erythroid transcriptional program as well as other key erythroid differentiation milestones including the reconfiguration of chromatin at erythroid gene loci, and the onset of erythropoietin dependence (1,2). Our published work shows that this switch takes place in early S phase of the last CFU-e generation, as cells transition from flow cytometric Subset 0 (S0, Lin-CD71medium) to Subset 1 (S1, Lin-CD71high). This S0/S1 commitment switch requires S phase progression, and unexpectedly, is associated with a 50% increase in the rate at which S phase cells synthesize DNA (1,2). This latter observation suggests that the duration of S phase is altered during the commitment switch, becoming shorter and faster in S phase cells in S1, compared with S phase cells in S0. We also found that the accelerated DNA synthesis in S phase cells in S1 is essential for an unusual process of genome-wide DNA demethylation, which is rate- limiting for erythroid gene transcription (2). While it is well documented that growth factors may promote shorter cell cycles, this has been considered to be largely the result of their ability to promote the transition from G1 to S phase, resulting in a shorter G1 phase. By contrast, relatively little is known of how S phase duration is modulated during cell fate decisions and differentiation. Here we determined directly the lengths of S phase in CFUe cells and during subsequent erythroid differentiation in the fetal liver in vivo; and identified the cyclin-dependent kinase (CDK) inhibitor, p57KIP2, as a key regulator of S phase duration at the S0/S1 commitment switch. To measure S phase duration, we injected pregnant female mice sequentially with two nucleotide analogs: first, with BrdU, and 2 hours later, with EdU. Fetal livers were harvested shortly following the second injection. Cells that were in S phase during the time of the first injection, but have left S phase by the time of the second injection, were measured as the BrdU+EdU- fraction. This approach allowed us to determine that S phase duration in S0 cells is 7.5 hours, transiently falling to under 4 hours in S1 cells, before resuming a slower pace in more differentiated, Ter119high erythroblasts. We identified the cyclin-dependent kinase (CDK) inhibitor, p57KIP2, as a novel negative regulator S phase DNA synthesis rate. p57KIP2 is expressed in S phase cells in S0, prior to the commitment switch, and is rapidly downregulated (>30 fold) during the switch (1). Here we found that its exogenous over-expression in S0 cells prevented S phase from becoming shorter and faster in S1. We therefore proceeded to investigate p57KIP2-null mice, found to die perinatally with a range of developmental abnormalities; their erythropoietic system was not investigated (3,4). We found that mouse embryos deficient for p57KIP2 are variably pale and/or anemic. Their fetal liver S0 cells showed premature shortening of S phase prior to the commitment switch, as deduced from an elevated DNA synthesis rate in S phase cells of p57KIP2+/- or p57KIP2-/- S0 cells (reaching 83% of the DNA synthesis rate in S1 cells in the same fetal livers; p We conclude that the efficient activation of the erythroid differentiation program at the S0/S1 commitment switch requires transient shortening of S phase. S phase becomes shorter and faster, most likely as a result of increased S phase CDK activation, when p57KIP2 expression is rapidly down-regulated at the S0/S1 transition. References: 1. Pop, R. et al., PLoS Biol, 2010. 8(9) 2. Shearstone, J.R., et al., Science, 2011. 334:799 3. Yan, Y., et al., G&D 1997. 11:973 4. Zhang, P., et al., Nature 1997. 387:151 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Signaling by the erythropoietin receptor (EpoR) is essential for the survival of definitive colony-forming unit-erythroid (CFU-e) progenitors and their erythroblast progeny. Here we used EpoR-null embryos to ask whether EpoR signaling might also exert essential non-survival functions in erythropoiesis. To address this, we rescued EpoR-null fetal liver cells from death by transducing them in vitro with either the anti-apoptotic protein Bcl-xL, or, as control, with the wild-type EpoR. The Bcl-xL-transduced EpoR-null cells survived, expressed hemoglobin and underwent morphological erythroid maturation and enucleation. However, unlike exogenous EpoR, exogenous Bcl-xL was unable to support the formation of EpoR-null CFU-e colonies in methylcellulose. The absence of colonies was explained by the finding that the Bcl-xL-transduced progenitors underwent fewer cell divisions than equivalent EpoR-transduced cells (1.1 vs. 2.9 in 24 hr, respectively) and had a slower rate of intra-S phase DNA synthesis, suggesting longer S phase duration. Multispectral imaging showed that the Bcl-xL-transduced cells matured prematurely, attaining smaller cell and nuclear size and a lower nuclear/cytoplasmic ratio at earlier time points than EpoR-transduced cells. Premature maturation was also evident by flow cytometric analysis. Thus, EpoR-null fetal liver cells in vivo arrest in their differentiation at the transition from subset S0 (Ter119-neg CD71-low) to S1 (Ter119-neg CD71-high) (Pop et al, PLoS Biology 2010). Rescue with EpoR in vitro allows EpoR-null progenitors to resume differentiation, sequentially upregulating CD71 and Ter119. By contrast, rescue of EpoR-null cells with Bcl-xL results in their premature upregulation of Ter119 and failure to upregulate CD71 to high levels. The cell cycle and differentiation deficits in Bcl-xL-supported, EpoR-null erythropoiesis were associated with a slower loss of DNA methylation from the erythroid genome, and with slower erythroid gene transcription. CD71 (the transferrin receptor) is a known target of EpoR and Stat5 signaling. We asked whether the deficits of EpoR-null erythropoiesis might be the result of low cell surface CD71 and the consequent reduced iron transport. In support of this hypothesis, we found that EpoR-null fetal liver cells that are transduced with both CD71 and Bcl-xL resume the normal maturation rate characteristic of EpoR-supported differentiation, as judged by multispectral imaging measurements of cell size and nuclear/cytoplasmic ratio. Further, we were able to restore rapid S phase to Bcl-xL-transduced EpoR-/- erythroblasts by culturing them in the presence of the cell-permeant iron chelator Fe-SIH (salicylaldehyde isonicotinoyl hydrazone), which is able to supply the cell interior with iron even in the absence of CD71 (Figure 1). We suggest that EpoR-mediated upregulation of CD71 at the onset of erythroid terminal differentiation determines the number and duration of erythroblast cell divisions by regulating iron homeostasis. Disclosures: No relevant conflicts of interest to declare.

Irreversible rapid cellular decisions are often controlled by network motifs known as bistable switches. We identified a cell-cycle regulated bistable switch that controls activation of the erythroid transcriptional program during early S phase of the last generation of erythroid colony-forming-unit progenitors (CFUe). This switch drives a rapid, multi-layered commitment event that activates GATA-1 transcription, renders the cells dependent on erythropoietin, and brings about chromatin reconfiguration at erythroid gene loci. In addition, it triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cell development. Approximately 25 to 30 percent of all methylation marks are lost from essentially all genomic elements during erythroid terminal differentiation. The bistable switch activating erythroid transcription consists of two linked double-negative feedback interactions of the erythroid transcriptional repressor PU.1, which antagonizes both S phase progression, and the erythroid master transcriptional regulator GATA-1. During operation of the switch, a rapid S phase-dependent decline in PU.1 activates GATA-1 transcription. The dependence of this switch on S phase progression coincides with a dramatic change in the nature of S phase itself, which becomes shorter and 50 percent faster. The accelerated intra-S phase DNA synthesis rate is essential for the loss of genome-wide DNA methylation, which in turn is required for the rapid induction of erythroid genes. Disclosures: No relevant conflicts of interest to declare.

The core binding factor (CBF) is a transcription factor that regulates key modulators of growth, survival and differentiation pathways. The CBF consists of a DNA binding α subunit (encoded by RUNX1, RUNX2, and RUNX3) and a common non-DNA binding β subunit (CBFB). RUNX1 and CBFB have been shown to be indispensible for embryo definitive hematopoiesis and to regulate adult hematopoiesis, and are targets of mutations in acute myeloid leukemia and myeloid dysplastic syndromes. We have shown that Runx2 is expressed in hematopoietic stem and early progenitor cells (HSPC: LSK+= Lin-ckit+Sca1+) and that it modulates leukemia latency in mice. However, little is known of Runx2 role in hematopoiesis. In this study, we have used conditional knock out mice for Runx1, Runx2, Runx1 and Runx2, and Cbfb (namely: Rx1ko, Rx2ko, Rx12dko, and Cbfbko) and the Cre deletors Mx1Cre and Vav-Cre, to show that Runx1 and Runx2 regulate hematopoietic lineage differentiation. Analysis of HSPCs 2 weeks post Mx1Cre induction, the HSCs (LSK+, FLT3-) were increased 4 fold in Rx1ko and Rx12dko mice, while the multipotential progenitors (MPPs:LSK+, FLT3+) of Rx12dko mice were expanded 5 fold. These data indicate that Runx1 regulates HSCs while both Runx factors regulate MPPs. The cell-intrinsic role of CBF factors in hematopoiesis was studied by evaluating the multilineage repopulation in competitive repopulation assay. To this end, recipient mice were transplanted 1:1 ratio of test (Rx1fl/fl, Rx2fl/fl, Rx1fl/flRx2fl/fl, or Cbfbfl/fl; each with Mx1Cre;CD45.2) and competitor (wt;CD45.1) bone marrow cells, treated with pIpC 4 weeks later, and analyzed every 4 weeks up to week 20 by flow cytometry. This analysis showed that Runx1 and Runx2 regulate differentiation in cell type specific manner. Runx1 and Runx2 have antagonistic functions in B cell lineage development, and Runx1 (but not Runx2) regulates T cell differentiation. The monocytes were not affected by the loss of Runx1 or Runx2, but were markedly reduced in the absence of both factors, suggesting that Runx1 and Runx2 may co-regulate monocyte development. The granulocytes (Mac1+Gr1+) were not affected in by Runx1 and/or Runx2, but were drastically reduced in Cbfb-null cells, suggesting that Runx3 could regulate granulocyte differentiation. The mechanism of HSPC regulation by Runx factors was studied by expression analysis of genes associated with HSC function. We have found that expression of adhesion molecules Alcam, Cx43 and Cxcr4 were deregulated in Rx1ko and Rx2ko HSCs and MPPs, as well as self-renewal factors, including Cdkn1a, Gfi1 and Mpl. To assess whether these alterations would impair the retention of HSPCs in the niche, we tested the ability of HSPCs to recover from cytotoxic stress, using 5-fluorouracil. At day 7, the percentage of immature (c-kit+) cells in peripheral blood had returned to normal in Rx1ko, Rx2ko, and wt mice. However, Rx12dko mice showed a 15-20 fold increase in circulating immature (c-kit+) cells. In addition, the administration of a second 5-fluorouracil dose at day 14 induced hematopoietic exhaustion and death in wt, Rx1ko and Rx2ko mice, but Rx12dko mice survived and recovered. These experiments indicate that loss of both Runx factors impairs the adhesion of HSCs to the niche and re-establishment of HSPC homeostasis To further study the role of CBF factors in hematopoiesis, we analyzed lineage contribution in Cbfbfl/fl, Vav-Cre mice at week 8 after birth. The HSPCs (LSKs) were increased 10 fold in Cbfb-null mice. These mice presented pancytopenia, with a 2-fold reduction in white blood cell count and anemia. The erythroid lineage was affected, including reduction of megakaryocyte/erythroid progenitors and Ter119+ progenitor cells in bone marrow, and reduction of red blood cell count and hematocrit in peripheral blood. The peripheral blood T and B cells were also reduced 6 and 2 fold respectively. In the myeloid compartment, the granulocyte/monocyte progenitor cells were increased 2 fold in bone marrow, and granulocytes increased 3 fold in peripheral blood. These studies reveal that Runx1 and Runx2 transcription factors regulate expression of adhesion and self-renewal genes in the HSPC compartment, modulating the homeostasis of HSCs in the bone marrow niche. In addition, Runx1 and Runx2 regulate hematopoiesis differentiation by synergistic and opposing effects in lineage specific manner. Disclosures: No relevant conflicts of interest to declare.

Abstract 2045 Signaling and transcriptional networks frequently contain negative autoregulatory feedback loops, where gene products negatively regulate their own induction or activation. These negative autoregulatory motifs are predicted to exert dual functions, accelerating gene induction, and providing stable gene expression levels in the face of the random perturbations inherent to biological systems. These predictions were confirmed experimentally in synthetic transcriptional circuits [1,2], but it is unknown whether they also hold in naturally occurring higher level biological networks. Here we studied the role of negative autoregulation by erythroid progenitors in the control of erythropoiesis. Erythropoietic rate, which may increase ten fold its basal rate during hypoxic stress, is dependent on the size of the erythroid progenitor pool, in turn regulated by the hormone erythropoietin (Epo). We recently found that, in addition, early erythroblasts negatively regulate their own numbers, through their co-expression of the death receptor Fas and its ligand FasL. Here we investigated the role of this negative autoregulation using Fas or FasL-deficient mice. We used the naturally-occuring mutant mouse strains, lpr and gld, deficient in Fas and FasL, respectively, back crossed onto the Rag1-/- mutant background, in order to avoid the autoimmune syndrome associated with Fas mutation. We proceeded to examine basal and stress erythropoiesis in the gld-Rag1-/- and lpr-Rag1-/- mice, and in matched Rag1-/- controls. We found that, in the basal steady state, the average size of the spleen early erythroid progenitor pool in gld-Rag1-/- and lpr-Rag1-/- mice increased 1.5 to 2 fold, consistent with loss of a negative regulator. Further, gld-Rag1-/- mice had a significantly elevated hematocrit in spite of normal Epo blood levels. The hematocrit was normal in the lpr-Rag1-/- mice, but Epo levels in this strain were significantly lower than normal. Taken together, these genetic mouse models show that Fas-mediated apoptosis of early erythroblasts in spleen negatively regulates erythropoietic rate in the basal state. We also found that the size of the progenitor pool was highly variable between individual Fas-deficient mice, suggesting reduced ability to maintain a stable steady-state erythorpoietic rate. In addition, gld-Rag1-/- and lpr-Rag1-/- mice had a significantly delayed erythropoietic stress response. Following an injection of a single dose of Epo (300 U/25 g), the early erythroblast population in spleen, ‘EryA’ (Ter119highCD71highFSChigh, [3]) expanded 30 to 60 fold its basal size. However, this expansion was significantly delayed in gld-Rag1-/- and lpr-Rag1-/- mice. Specifically, on day 2 of the stress response, control Rag1-/- mice had a 30% larger EryA progenitor pool compared with lpr-Rag1-/- mice, a difference equivalent to 10 fold the size of the basal EryA pool. Consequently, control mice achieved a higher hematocrit 24 hours earlier than mutant gld-Rag1-/- and lpr-Rag1-/- mice. We propose that the larger expansion of EryA cells during the stress response in control mice is due to the recruitment of a reserve population of Fas-positive EryA. This reserve population, absent in mice deficient in the Fas pathway, undergoes Fas-mediated apoptosis in the basal steady state. However, high Epo levels during the stress response suppress Fas expression [3], rescuing these cells from apoptosis and accelerating the stress response. These findings show, using genetic mouse models, that the stability of stead-state erythropoietic rate and its rapid stress response are key outcomes of negative autoregulation within the erythroid progenitor pool. Furthermore, they show experimentally that dynamic properties of negative autoregulatory loops in simple low-level networks are also exerted in the context of complex inter-cellular, tissue level networks such as those that regulate erythroipoietic rate. References: 1. Becskei A, Serrano L (2000) Engineering stability in gene networks by autoregulation. Nature 405: 590–593. 2. Rosenfeld N, Elowitz MB, Alon U (2002) Negative autoregulation speeds the response times of transcription networks. J Mol Biol 323: 785–793. 3. Liu Y, Pop R, Sadegh C, Brugnara C, Haase VH, et al. (2006) Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood 108: 123–133. Disclosures: No relevant conflicts of interest to declare.

Abstract 2083 In the mammalian genome cytosine residues that are followed by guanine (5’-CpG-3’ dinucleotides) are frequently methylated, a modification that is associated with transcriptional silencing. Two genome-wide waves of demethylation, in primordial germ cells and in the early pre-implantation embryo, erase methylation marks and are each followed by de novo methylation, setting up a pattern subsequently inherited throughout development [1]. While no global methylation changes are thought to occur during further somatic development, methylation does alter at gene-specific loci, contributing to tissue-specific patterns of gene expression. We set out to study dynamic changes in DNA methylation during erythropoiesis. We used flow cytometry and the cell surface markers CD71 and Ter119 to subdivide freshly isolated fetal liver cells into a developmental sequence of six subsets, from the least mature Subset 0 (S0), to the most mature Subset 5 (S5) [2]. We measured DNA methylation in genomic DNA prepared from freshly sorted S0 to S5 cells. Surprisingly, we found that demethylation at the erythroid-specific β-globin locus control region (LCR) was coincident with progressive genome-wide methylation loss. Both global demethylation as well as demethylation at the β-globin LCR began with the upregulation of CD71 at the onset of erythroid terminal differentiation, and continued with erythroid maturation, with global hypomethylation persisting during enucleation. We employed several distinct methodologies to measure global DNA methylation level. Using Enzyme-Linked Immunosorbent Assay (ELISA), we found that genomic DNA isolated from increasingly mature erythroblasts had progressively reduced binding to a 5-methylcytosine-specific antibody. We also used the LUminometric Methylation Assay (LUMA) to compare the genome-wide cleavage of CCGG sites by each of the isoschizomers HpaII and MspI, which are methylation sensitive and insensitive, respectively. Both the ELISA and LUMA assays showed a global, progressive and significant loss of DNA methylation with erythroid differentiation: 70% of CpG dinucleotides genome-wide were methylated in S0, decreasing to 40–50% by S4/5 (p To characterize the global loss in methylation further, we examined the status of imprinted genes and of repetitive transposable elements, since both represent genetic loci that are usually stably and highly methylated in somatic cells. We found loss of methylation in imprinted loci, including PEG3 and the H19 Differentially Methylated Region (DMR). We also found a significant loss of methylation at the Long Interspersed Nuclear Element (LINE-1), a repetitive retrotransposon, whose methylation level decreased from over 90% in S0 cells, to 70% in S4/5. Mechanistically, global demethylation was associated with a rapid decline in the DNA methyltransferases DNMT3a and DNMT3b. However, exogenous re-expression of these enzymes in vitro was not sufficient to reverse the process. Both global and erythroid-specific demethylation required rapid DNA replication, triggered with the onset of erythroid terminal differentiation. We were able to slow down demethylation quantitatively by slowing down the rate of DNA replication with aphidicolin, an inhibitor of DNA polymerase α. Global loss of DNA methylation was not associated with a global increase in transcription, as determined by GeneChip analysis, nor was it associated with increased transcription of the LINE-1 retrotransposon. We propose that global demethylation is a consequence of global cellular mechanisms required for the rapid demethylation and induction of β-globin and other erythroid genes. Our findings suggest mechanisms of global demethylation in development and disease, and show that contrary to previously held dogma, DNA demethylation occurs globally during physiological somatic cell differentiation. References: 1. Reik W, Dean W, Walter J (2001) Epigenetic reprogramming in mammalian development. Science 293: 1089–1093. 2. Socolovsky M, Murrell M, Liu Y, Pop R, Porpiglia E, et al. (2007) Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis. PLoS Biol 5: e252. Disclosures: No relevant conflicts of interest to declare.

Erythropoietic rate varies through a large dynamic range. Its principal regulator is the hormone erythropoietin (Epo), which, in response to hypoxic stress increases up to 1000 fold its basal level, driving erythropoietic rate by up to ten fold. The mechanisms in erythroid progenitors that regulate large, rapid and yet precise changes in erythropoietic rate are not yet understood. It’s been suggested that survival pathways activated by the Epo receptor (EpoR) underlie its regulation of erythropoietic rate. Studies of cultured erythroid cells have identified several anti-apoptotic regulators as EpoR targets. However, their potential contribution to erythropoietic rate in vivo had not been investigated. Here we assessed the in-vivo role of two EpoR-activated survival pathways: EpoR induction of the anti-apoptotic regulator bcl-xL, and EpoR-mediated suppression of erythroblast Fas and FasL expression. We found that these pathways differ markedly in their regulation of erythropoietic rate. We used flow-cytometric measurement of bclxL, Fas and FasL in each of four erythroblast subsets of increasing maturity (Liu et al. Blood 2006), ProE (Ter119medCD71highFSChigh), EryA (Ter119highCD71highFSChigh), EryB (Ter119highCD71highFSClow) and EryC (Ter119highCD71lowFSClow). Acute erythropoietic stress was induced by Epo injection or by subjecting mice to reduced atmospheric oxygen. Measurements were made on freshly explanted mouse bone-marrow and spleen, either in the basal state or at different time points following induction of stress. Acute erythropoietic stress caused a rapid but transient induction of bcl-xL that peaked at 12 to 18 hours, principally in splenic ProE and EryA. Bcl-xL levels returned to baseline by 24 hours, before resolution of stress. A similar time course was found for induction of the bcl-xL mRNA. In contrast to the acute response, in mouse models of chronic erythropoietic stress, including anemic mice with beta thalassemia, bcl-xL was not increased above baseline. However, an acute Epo injection in these mice caused transient bcl-xL induction similar to that seen in healthy mice. The magnitude of bcl-xL induction in acute stress was similar, regardless of the absolute change in Epo concentration. We conclude that EpoR-mediated bcl-xL induction is designed to detect a rapid change in Epo, rather than the absolute level of Epo concentration. It undergoes rapid adaptation, and in both these properties is reminiscent of sensory pathways or bacterial chemotaxis. We suggest this pathway provides a ‘stop-gap’ that enhances erythroblast survival until slower but more permanent pathways are activated. EpoR signaling also causes suppression of erythroblast Fas and FasL, which are co-expressed in splenic ProE and EryA. The size of the EryA subset increases with erythropoietic stress over a wide range. We found that Epo-mediated suppression of Fas/FasL is inversely related to the size of the EryA subset, regardless of whether erythropoietic stress is acute or chronic. Therefore, unlike bcl-xL induction, EpoR-mediated suppression of Fas/FasL does not undergo adaptation, is a function of the absolute degree of stress and Epo concentration, and likely responsible for long-term maintenance of EryA subset size. To investigate this further, we generated mice deficient in Fas (lpr) or FasL (gld) on an immune deficient background (rag1−/−) in order to circumvent the autoimmune syndrome of lpr and gld mice. Both these mouse strains showed a significant increase in their CFU-e, ProE and EryA subsets, particularly in spleen, and the gld/rag1−/− strain also showed increased basal hematocrit. This confirms a negative regulatory effect for Fas in erythropoiesis. Of note, we also found a striking increase in variance for the size of each of these subsets in the mutant mice. We conclude that, in addition to determining the size of the EryA and other erythroid precursor subsets appropriate for each stress level, the Fas-FasL interaction provides a stabilizing mechanism that filters out inappropriate variation in the number of CFU-e, ProE and EryA subsets and in erythropoietic rate. Taken together, our studies in vivo elicited system-level functions for two survival pathways which were not apparent from their investigation in vitro. In combination, these pathways endow the erythropoietic system with a fast response time and with robustness against inappropriate fluctuations in erythropoietic rate.

The essential role of glycoprotein hormone erythropoietin (Epo) and its receptor, EpoR, in erythroid development is well established: both the EpoR−/− and Epo−/− mouse embryos die on embryonic day 13 (E13) due to failure of definitive erythropoiesis in fetal liver (Wu et al. 1995). It has been suggested that Epo’s principal role during erythropoiesis is to protect erythroid progenitors from apoptosis (Koury and Bondurant, Science 1990). Bcl-xL, an anti-apoptotic member of the bcl-2 family, is induced by EpoR signaling in erythroid cells via the Jak2/Stat5 pathway (Silva et al., Blood 1996; Socolovsky et al., Cell, 1999). Bcl-xL is essential for erythroid maturation: bcl-xL−/− embryos die in utero at the same stage as as EpoR−/− mice, lacking definitive erythropoiesis (Motoyama et al., Science 1995; J Exp Med, 1999). Recenlty, it has been shown that over-expression of bcl-xL in primary wild-type erythroblasts confers Epo independence on these cells in vitro and allows them to complete their differentiaion into red blood cells (Dolznig et al., Curr Biol, 2002). Here we reasoned that if the principal function of EpoR signaling is suppression of apoptosis via bcl-xL, it should be possible to rescue all aspects of erythroid differentiation in EpoR−/− fetal liver progenitors by retrovirally-transducing these cells with bcl-xL. We infected EpoR−/− fetal liver progenitors with bicistronic retroviral vectors expressing either bcl-xL or EpoR, each linked via an IRES sequence to a GFP reporter. Control EpoR−/− cells were infected with ‘empty’ bicistronic vector. Infection rates were in excess of 30% for all constructs, and transduced cells were identified for further analysis using GFP fluorescence. We examined terminal differentiation of the transduced EpoR−/− cells over the ensuing 48 hours, using several distinct assays, including their expression of the cell-surface differentiation markers CD71 and Ter119 by FACS, their ability to give rise to CFU-e colonies in semi-solid medium, their cell-cycle status using DNA content analysis and BrdU incorporation, and their maturation and hemoglobinization by diaminobenzidine staining and light microscopy. We found that EpoR−/− progenitors transduced with bcl-xL were protected from apoptosis, and underwent morphological changes characteristic of erythroid maturation, including decreasing cell size, nuclear condensation and expulsion, and accumulation of hemoglobin. These cells also upregulated the erythroid-specific cell surface marker Ter119. However, unlike EpoR−/− cells transduced with EpoR, bcl-xL -transduced cells did not express high levels of CD71, and failed to give rise to CFU-e colonies in semi-solid medium. Instead, they gave rise to small colonies of 6 cells or less. Cell cycle analysis showed that, throughout the 48 hours of erythroid terminal differentiation, the population of bcl-xL-transduced EpoR−/− cells had a lower fraction of cells in S-phase than control, EpoR-transduced EpoR−/− cells. The cell-cycle status of control, terminally-differentiating wild-type erythroid fetal liver progenitors was not altered by transduction with bcl-xL, excluding the possibility that it directly inhibits S-phase. Taken together our results indicate that bcl-xL does not rescue all aspects of erythroid differentiation in EpoR−/− erythroid progenitors. Specifically, the proliferative program during erythroid terminal differentiation is directly dependent on EpoR signaling, and is not simply a default pathway secondary to EpoR’s anti-apoptotic effect.

The rate of red blood cell production increases up to ten-fold during stress erythropoiesis. We have recently identified stress-responsive CD71highTer119positive early erythroblast subsets in freshly-isolated mouse hematopoietic tissue by flow cytometry. Both the absolute number and relative frequency of these early erythroblast subsets increase dramatically during stress. We have shown that this erythroblast expansion is associated with enhanced erythroblast viability, which is at least in part due to down-regulation of the death-receptor Fas, and its ligand, FasL from early erythroblasts by erythropoietin-receptor (EpoR) signaling (Liu et al., Blood 2006). The anti-apoptotic protein bcl-xL is induced in differentiating erythroid cells in vitro by EpoR and Stat5 signaling (Socolovsky et al., Cell 1999). Bcl-xL is essential for erythroid cell viability and is required for the maintenance of the normal basal hematocrit (Motoyama et al., Science 1995). However, it is unclear whether bcl-xL plays a role in enhancing erythroblast viability during the stress response. Serum factors other than Epo may modulate erythroid bcl-xL levels (Dolznig et al., Oncogene 2006), complicating the interpretation of bcl-xL measurements in cultured erythroid cells in vitro. Therefore, we examined the potential role of bcl-xL in stress erythropoiesis by measuring bcl-xL mRNA directly in CD71highTer119positive early erythroblasts in vivo in a mouse model of stress. We mimicked the effect of acute erythropoietic stress by injecting adult Balb/C mice with a single dose of Epo (50 mg/kg subcutaneously). Control mice were injected with an equal volume of saline. Spleen cells were harvested at 3, 16, 24, 48 and 72 hours post injection, and CD71highTer119positive early erythroblasts were immediately sorted by flow-cytometry. RNA was extracted from these freshly sorted cells and used in quantitative real-time PCR to measure bcl-xL mRNA expression. We normalized the level of bcl-xL mRNA in each sample by expressing it relative to beta-actin mRNA. At least 3 independent experiments were conducted for each time point. In parallel, we measured serum Epo concentration following Epo injection by ELISA. This showed that Epo increased approximately 100 fold by 40 minutes post-injection, reaching a peak by 6 hours and returning to basline levels by 48 hours. We found that bcl-xL mRNA began to increase in spleen early erythroblasts by 3 hours following Epo injection. By 16 hours, bcl-xL mRNA in Epo-injected mice was three-fold higher than in mice injected with saline. Bcl-xL mRNA continued to be elevated, by 2.5 fold, at 24 hours, but declined back to baseline levels by 48 hours. The time course of the increase in splenic early erythroblast bcl-xL mRNA therefore closely parallels the time course of serum Epo. The induction of early erythroblat bcl-xL mRNA suggests it is likely to contribute to the viability of stress-responsive CD71highTer119positive early erythroblasts, and therefore to the increased erythropoietic rate during the stress response.

Tissue hypoxia triggers the erythropoietic stress response, where high blood erythropoietin (Epo) stimulates increased red blood cell production rate. Stat5 is rapidly phosphorylated following ligation of the Epo receptor (EpoR) in erythroid cells in vitro and is required for normal erythropoiesis [Socolovsky et al. Blood, 2001; Cui et al., Mol Cel Biol, 2004]. Stat5-deficient mice, and mice expressing a truncated EpoR lacking Stat5 docking sites, are impaired in their response to erythropoietic stress, suggesting thatStat5 mediates EpoR signaling during stress [Socolovsky et al., 2001; Menon et al., J Clin Invest, 2006]. The identity of the erythroid progenitors in which Stat5 becomes active during stress, and the time-course of its activation, are not known. Recently, we developed flow-cytometric techniques that identify stress-responsive erythroblast subsets directly in freshly-explanted mouse hematopoietic tissue [Liu et al., Blood 2006]. Here we combined these techniques with intracellular flow-cytometry [Krutzik et al, J Immunol., 2005], to measure Stat5 activation within early erythroblasts in vivo. We mimicked the effects of acute erythropoietic stress by injecting adult Balb/C mice with a single dose of Epo (10 IU/gram sub-cutaneously), and harvested spleen and bone marrow at different time points following Epo injection. These cells were labeled for the cell-surface markers Ter119 and CD71, and intracellularly with a specfic antibody against phospho-Stat5. Serum Epo was measured by ELISA. Baseline Epo (10 to 50 mU/ml) increased to 600 mU/ml by 10 minutes post injection, peaked by 6 hours and remained high (over 5000 mU/ml) for 24 hours. Stat5 phosphorylation (=phospho-Stat5) was apparent by 15 minutes in both bone-marrow and spleen. In both tissues, it was highest in the least differentiated, ProE and Ery.A erythroblasts (Ter119-med CD71-high, and Ter119-high CD71-high FSC-high, respectively, Liu et al. 2006). In bone-marrow, the percentage of ProE that were positive for phospho-Stat5 (phospho-Stat5+) increased from a baseline of less than 1% to 65% by 30 minutes, but declined to 10% of ProE by 6 hours. This low-level of phospho-Stat5+ cells was maintained for the ensuing 10 hours. Of interest, in spite of the large variations in the percent of phospho-Stat5+ cells, the median phospho-Stat5 signal remained constant within the phospho-Stat5+ erythroblasts. This suggests that erythroblasts are either ‘on’ or ‘off’ with respect to Stat5 activation, and that the principal variant is the fraction of cells that are ‘on’ in the tissue. The decline in phospho-Stat5+ cells by 6 hours occurred in spite of persisting, high serum Epo, suggesting the activation of negative feedback mechanisms that limit EpoR signaling. We also noted a clear difference in the sensitivity of otherwise similar erythroblast subsets between spleen and bone-marrow: in spleen, a smaller percentage of erythroblasts became phospho-Stat5+, the signal was slower to develop and diminished sooner than in bone-marrow. We conclude that Stat5 phosphorylation occurs rapidly upon an increase in serum Epo, but is likely to be damped from its peak by negative feedback meachanisms. Spleen erythroblasts are less sensitive than bone-marrow erythroblasts to Epo activation. Further, the principal regulation of the phospho-Stat5 signal appears to be at the level of the tissue, where the main variable is the fraction of cells expressing phospho-Stat5, rather than the level of phospho-Stat5 per cell. The molecular mechanisms responsible for this type of regulation remain to be elucidated.

The stem cell leukaemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in normal haematopoiesis and angiogenesis. The SCL gene is normally expressed in haematopoietic stem cells, mast cells, megakaryocytes, endothelium and smooth muscle. Aberrant expression of the SCL gene leads to T-cell acute lymphoblastic leukaemia, whereas SCL−/− mice die due to the absence of haematopoiesis. Hence, temporal and spatial regulation of SCL expression is essential. Our laboratory has previously characterised a 5.5 kb enhancer located 3′ of the SCL transcription start site, which is capable of targeting expression of b-galactozidase (LacZ) reporter gene to haematopoietic stem cells in the foetal liver and the bone marrow, as well as embryonic endothelium. Subsequent experiments showed that a 641-base pair core enhancer gave an identical pattern of lacZ expression in the embryo. However, it was unclear if the same element (later referred to as +19 core enhancer) was capable of maintaining reporter gene expression into the adulthood, since no lacZ activity was observed in postnatal mice. Using a transgenic construct containing a eukaryotic reporter gene, human placental alkaline phosphatase, we show that in the haematopoietic system, the +19 core enhancer is sufficient to target foetal liver and bone marrow HSCs, as well as mast cells and megakaryocytes. In the erythroid lineage, the enhancer is active only during the earlier stages of erythropoiesis, despite high level of SCL expression throughout erythroblast maturation, suggesting that an additional element is likely to be required to maintain SCL expression. The enhancer also targets embryonic and adult endothelium, together with vascular and visceral smooth muscle. Taken together, our results demonstrate that the 641-bp +19 core enhancer is sufficient to integrate signals upstream of SCL in blood, endothelium and smooth muscle. Our data also suggest that developmental relationship between these three mesoderm-derived lineages could be defined through a common transcriptional environment, and indicate that SCL may play a wider role in mesodermal development than previously thought.