ALBERT

Description: Effective teamwork in an initially leaderless group requires a high level of collective leadership emerging from dynamic interactions among group members. Leader emergence is a crucial topic in collective leadership, yet it is challenging to investigate as the problem context is typically highly complex and dynamic. Here, we explore leadership emergence and leadership perception by means of computational simulations whose assumptions and parameters were informed by empirical research and human-subject experiments. Our agent-based model describes the process of group planning. Each agent is assigned with three key attributes: talkativeness, intelligence, and credibility. An agent can propose a suggestion to modify the group plan as a speaker or respond and evaluate others’ suggestions and leadership as a listener. Simulation results suggested that agents with high values of talkativeness, intelligence, and credibility tended to be perceived as leaders by their peers. Results also showed that talkativeness may be the most significant and instantaneous predictor for leader emergence of the three investigated attributes: talkativeness, intelligence, and credibility. In terms of group performance, smaller groups may outperform larger groups regarding their problem-solving ability in the beginning, but their performance tends to be of no significant difference in a long run. These results match the empirical literature and offer a mechanistic, operationalized description of the collective leadership processes.

Description: Abstract 3442 The maturation of a committed erythroid progenitor to a functional red blood cell is a complex process involving significant changes in gene expression during a time of rapid cell division and nuclear condensation. LSD1 (Lysine-Specific Histone Demethylase 1) is a histone H3 lysine 4 (H3K4) and lysine 9 (H3K9) demethylase that plays pivotal role in this process. LSD1 participates in both enhancer and repressor complexes, and facilitates repression of γ-globin by participating in the Direct Repeat Erythroid Complex (Cui, MCB, 2011). LSD1 inhibitors Tranylcypromine (TCP) and Pargyline (PG) are being investigated as potential therapies for the β-globinopathies, however little is known about the broader functional or genomic consequences of LSD1 inhibition on terminal erythroid maturation. Both TCP and PG impair erythroid maturation in Extensively Self Renewing Erythroblasts (ESREs), a primary cell model of terminal erythroid maturation. ESREs are primary cells derived from fetal liver that proliferate extensively in culture, but retain the ability to appropriately mature and enucleate (England, Blood, 2011), making them ideal for functional and genomic studies of terminal erythroid maturation. In untreated or vehicle (DMSO) treated cultures 〉90% of cells are benzidine positive by day3 of maturation. In contrast, cultures treated with 400um PG, 1um TCP, or 2um TCP were 72, 42, and 33% benzidine positive by maturation day3, respectively. Cells in the TCP-and PG- treated cultures also had morphologic evidence of impaired maturation, with larger nuclei and more basophilic cytoplasm. In addition to its role as a histone demethylase, LSD1 stabilizes DNMT1 (DNA methyltransferase 1; Wang, Nat Genet 2009). We hypothesized that loss of DNA methylation contributes to the maturation impairment seen with LSD1 inhibitors, and that inhibition of DNMTs with decitabine would also impair terminal erythroid maturation. Consistent with this hypothesis, ESREs treated with decitabine demonstrated a dose-dependent impairment of maturation similar to that seen with PG and TCP. To elucidate the molecular mechanisms underlying the maturation impairment in TCP- and PG- treated cultures, levels of H3K4me2 and methylated DNA (5-methyl cytosine, 5-mC) were assessed both globally and at specific loci. An ELISA (Enzyme-linked Immunosorbent Assay) was used to assess global levels of H3K4me2 and 5-mC in vehicle-, PG-, and TCP-treated cultures after 24 hours of maturation. Global levels of H3K4me2 were significantly higher in PG- and TCP- treated samples than control. In maturing cells, there was no significant difference in the level of 5-mC in vehicle- and inhibitor- treated cultures. It is well established, however, that global DNA methylation decreases with erythroid maturation (Seashore, Science, 2011), and a significant decrease in 5-mC occurs in ESREs during the first 24hrs of maturation. As TCP- and vehicle- treated cultures mature differently, the effect of TCP on 5-mC levels was also assessed in self-renewing ESREs at the proerythroblast stage. Unlike maturing cells, TCP-treated proerythroblasts had a significant decrease in 5-mC levels compared to control. Chromatin immunoprecipitation (ChIP) was used to examine the local effects of LSD1 inhibition on H3K4me2 enrichment at erythroid-specific promoters. TCP-treated cultures had non-uniform changes in H3K4me2 enrichment, with levels increased at some promoters (e.g. protein 4.1,εy-globin), but unchanged at others (e.g. β-globin). To further study the relationship between LSD1 inhibition and H3K4me2 levels, ChIP-seq was used to identify LSD1 sites that co-localized with putative enhancers, defined as peaks of H3K4me2 binding 〉 than 1kb from a transcription start site. ChIP-qPCR was used to compare the level of H3K4me2 at 5 validated enhancer-associated LSD1 sites in vehicle- and TCP-treated cells. The effect of TCP was variable, with only 2/5 enhancer-associated LSD1 sites having increased H3K4me2. Lastly, the local effects of inhibitors on 5-mC were examined using a methyl binding domain pulldown coupled with qPCR. In TCP-treated cells, 5-mC levels declined at several loci, most notably at the εy-globin promoter. Taken together, these results suggest that the impaired erythroid maturation associated with LSD1 inhibition results from the perturbation of multiple mechanisms of epigenetic regulation. Disclosures: No relevant conflicts of interest to declare.

Description: The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (〉100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite 〉1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.

Description: Abstract 2383 Erythropoietin (EPO) acting through its receptor (EPOR), is a critical regulator of definitive erythropoiesis. The EPOR signaling network has been extensively studied in cell lines overexpressing various forms of EPOR. Together with genetic models, these studies have defined an EPOR-JAK2-STAT5 pathway that is critical for the formation of red blood cells. However, the hematopoietic phenotype of mice with a truncated EPOR incapable of activating STAT5 (EPOR-HM) suggests that STAT5 activation is not the only pathway mediating the erythropoietic effects of EPO-EPOR signaling. Our recent studies indicate that EPO-EPOR signaling also regulates primitive erythroblast maturation in the mammalian embryo. To better understand EPOR signaling in the context of primary erythroid cells, we utilized primitive erythroblasts from wild-type and EPOR-HM murine embryos. Robust phosphorylation of STAT5 was detected by western blot in freshly isolated primitive erythroblasts from wild-type embryos. As expected, there is no STAT5 phosphorylation evident in primitive erythroblasts from EPOR-HM embryos. Surprisingly, EPOR-HM embryos produce nearly normal numbers of primitive erythroblasts and we detected equivalent induction of STAT target genes following EPO stimulation in wild-type and EPOR-HM primitive erythroblasts. To address the discrepancy between these data and the accepted model of EPOR-STAT5 signaling, we searched for compensatory signaling via other STAT family members. We found that STAT3, but not STAT1, was phosphorylated in primitive erythroblasts from EPOR-HM embryos, suggesting that phospho-STAT3 compensates for the loss of phospho-STAT5 in the EPOR-HM mice. We also detected a lower level of STAT3 phosphorylation in wild-type primitive erythroblasts that was increased upon in vitro EPO stimulation. These results indicate that EPOR-STAT3 signaling normally occurs in primitive erythroblasts. Furthermore, our preliminary data show that STAT3 transcript levels are higher in primitive erythroblasts compared to definitive erythroblasts from the fetal liver or bone marrow, suggesting that STAT3 activation during erythropoiesis may be lineage-dependent. To test this hypothesis, we utilized a small molecule inhibitor of STAT3 to examine the function of STAT3 signaling both in primitive and in definitive erythroblasts during in vitro maturation. The terminal maturation of definitive erythroblasts is unaffected by STAT3 inhibition. However, inhibition of STAT3 causes a 40% reduction in primitive erythroblast proliferation in a novel 2-step ex vivo culture system. We conclude that, in contrast to definitive erythroblasts, STAT3 is activated downstream of EPOR in primitive erythroid cells. EPOR-STAT3 signaling has been reported in non-erythroid cells as well as in several cancer cell types. Therefore the study of EPOR signaling in primary embryonic erythroblasts may provide novel insights regarding the function of EPO in these cells. Disclosures: No relevant conflicts of interest to declare.

Description: In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (102- to 105-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (106- to 1060-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.