Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

mTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

Vasoactive-Inotropic Score as a Determinant of Timely Initiation of Venoarterial Extracorporeal Membrane Oxygenation in Patients With Cardiogenic Shock.

BACKGROUND: The predictive role of the vasoactive-inotropic score (VIS) for clinical outcomes after venoarterial extracorporeal membrane oxygenation (VA-ECMO) in patients with cardiogenic shock is not well known. This study investigated the predictive value of VIS on in-hospital outcomes and the determination of optimal timing for the initiation of VA-ECMO.Methods and Results:Overall, 160 patients with cardiogenic shock requiring VA-ECMO who were treated between December 2012 and August 2018 were analyzed. The in-hospital outcomes according to VIS were compared. Pre-ECMO VIS had an area under the receiver-operating characteristic curve (AUC) of 0.60 (P=0.03) for the prediction of in-hospital death. When the patients were divided into the high (≥32) and low (<32) VIS groups, the high VIS group had a higher rate of in-hospital death (P=0.002) and a lower rate of ECMO weaning (P=0.004). The difference in in-hospital death according to VIS was significant only in patients with a cardiogenic shock of non-ischemic etiology (P=0.01). Extracorporeal cardiopulmonary resuscitation (hazard ratio [HR], 1.99), age (HR, 1.02), pre-ECMO lactate (HR, 1.06), and VIS ≥32 (HR, 2.46) were independently predictive of in-hospital death. CONCLUSIONS: Among patients with cardiogenic shock requiring VA-ECMO, the initiation of VA-ECMO before reaching high VIS (≥32) showed better in-hospital outcomes, suggesting that VIS may be a potential marker for determining the initiation of hemodynamic support with VA-ECMO.

Impact of Event Scale-6 (IES-6) for U.S. adults who experienced the COVID-19 pandemic.

BACKGROUND: COVID-19 pandemic causes psychological problems such as stress. It is important to accurately identify the level of stress and establish effective intervention. The Impact of Event Scale-6 (IES-6) is widely used for post-traumatic stress disorder (PTSD) screening by measuring the level of subjective stress, but there has been no research on its psychometric properties with individuals who experienced the COVID-19 pandemic. METHODS: A random sample of 600 participants were randomly selected from a COVID-19 survey database (n = 6391). Rasch analysis was conducted to examine item fit, rating scale structure, construct validity, differential item functioning (DIF), and precision of the IES-6. RESULTS: The principal component analysis of Rasch residuals (54.1% of the raw variance explained) and the average of residual correlations (average r = .19) supported the unidimensionality structure in the IES-6. The rating scale was suitable, and the item difficulty hierarchy was logical. The item fit and the DIF contrast were acceptable, except for item 5. The IES-6's person reliability was .76, which was also an acceptable level. CONCLUSIONS: This study showed that the IES-6 has acceptable item-level psychometrics for screening the stress level in adults in the United States for individuals who have experienced the COVID-19 pandemic. The findings suggested that the IES-6 would be useful for the rapid identification of the high-level stressand allow clinicians to quickly provide interventions for people with the COVID-19 related stress and their families.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Outcomes of left ventricular unloading with a transseptal cannula during extracorporeal membrane oxygenation in adults.

We evaluated the benefit of left ventricular (LV) unloading using a percutaneous transseptal left atrial (LA) drain catheter via femoral vein incorporated into the ECMO venous circuit. This single-center retrospective observational study analyzed clinical outcomes of the LA venting group (N = 62) who underwent percutaneous transseptal LA drain placement comparing with the conventionally treated control group (N = 62) with an arterial pulse pressure below 10 mm Hg for at least 24 hours from December 2012 to August 2018. The ECMO weaning rate (61.3% vs. 38.7%, P = .012) and cardiac transplantation rate (29.0% vs. 11.3%, P = .014) were higher in the LA venting group than in the control group. Inhospital mortality was not significantly different (56.5% vs. 69.4%, P = .191). Pulmonary congestion mostly improved after LA decompression (61.3%, P = .003). A serum lactate level at 24 hours after LA venting of more than 2.2 mmol/L was associated with poor outcomes. LA venting via transseptal cannula reduced pulmonary venous congestion and achieved higher rates of successful ECMO weaning and cardiac transplantation. Placement of a transseptal venous drain cannula should be considered in patients with uncontrolled pulmonary edema secondary to severe LV loading undergoing VA-ECMO.

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

Protective role of endogenous plasmalogens against hepatic steatosis and steatohepatitis in mice.

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat; the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of FC in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing sterol regulatory element-binding protein 2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat small interfering RNA treatment significantly decreased peroxisome proliferator-activated receptor α (Pparα) expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor, alkyl glycerol (AG), prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat+/- mice were more susceptible to hepatic lipid accumulation and less responsive to the preventive effect of fluvastatin on NASH development, suggesting that endogenous plasmalogens prevent hepatic steatosis and NASH. CONCLUSION: Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis through GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH. (Hepatology 2017;66:416-431).

Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder (Paralichthys olivaceus).

The immune tone is defined as an immunological state during which the readiness for immune response is potentiated. The establishment of immune tone in the gut of olive flounder (Paralichthys olivaceus) was investigated by feeding Lactococcus lactis BFE920 (LL) or Lactobacillus plantarum FGL0001 (LP). LL-fed flounder showed significantly increased levels of regulatory genes (FOXP3, IL-10, and TGF-ß1), CD18, and CD83 in the gut. In contrast, LP feeding drastically increased proinflammatory genes (T-bet, IL-1ß, and IFN-γ) and CD18. This indicates that LL and LP establish different types of local immune tones in the gut through differential activation of innate immune cells: LL activates both macrophages and dendritic cells while LP activates macrophages only. Both of the immune tones required at least a total of 6 probiotic feeds during 72 h for a stable establishment. Once established, the type of immune tone remained steady even up to 30 days (a total of 60 feeds) probiotics feeding. The LL-induced regulatory immune tone enhanced the level of occludin, a tight junction molecule, significantly more than that observed with the proinflammatory immune tone established by LP feeding. Consequently, LL-fed fish showed considerably lower gut permeability than that of the LP-fed group. Furthermore, when orally challenged by Edwardsiella tarda, LL-fed flounder survived at a significantly higher rate than LP-fed fish. The data clearly demonstrate that individual probiotics establish distinct types of immune tone in the fish gut, which in turn influences the immunological status as well as the physiology of the gut. Selection of proper probiotics may be essential for optimal effects in aquaculture farming.

Rearrangements of 2.5 kilobases of noncoding DNA from the Drosophila even-skipped locus define predictive rules of genomic cis-regulatory logic.

Rearrangements of about 2.5 kilobases of regulatory DNA located 5' of the transcription start site of the Drosophila even-skipped locus generate large-scale changes in the expression of even-skipped stripes 2, 3, and 7. The most radical effects are generated by juxtaposing the minimal stripe enhancers MSE2 and MSE3 for stripes 2 and 3 with and without small "spacer" segments less than 360 bp in length. We placed these fusion constructs in a targeted transformation site and obtained quantitative expression data for these transformants together with their controlling transcription factors at cellular resolution. These data demonstrated that the rearrangements can alter expression levels in stripe 2 and the 2-3 interstripe by a factor of more than 10. We reasoned that this behavior would place tight constraints on possible rules of genomic cis-regulatory logic. To find these constraints, we confronted our new expression data together with previously obtained data on other constructs with a computational model. The model contained representations of thermodynamic protein-DNA interactions including steric interference and cooperative binding, short-range repression, direct repression, activation, and coactivation. The model was highly constrained by the training data, which it described within the limits of experimental error. The model, so constrained, was able to correctly predict expression patterns driven by enhancers for other Drosophila genes; even-skipped enhancers not included in the training set; stripe 2, 3, and 7 enhancers from various Drosophilid and Sepsid species; and long segments of even-skipped regulatory DNA that contain multiple enhancers. The model further demonstrated that elevated expression driven by a fusion of MSE2 and MSE3 was a consequence of the recruitment of a portion of MSE3 to become a functional component of MSE2, demonstrating that cis-regulatory "elements" are not elementary objects.

Ancestral resurrection of the Drosophila S2E enhancer reveals accessible evolutionary paths through compensatory change.

Upstream regulatory sequences that control gene expression evolve rapidly, yet the expression patterns and functions of most genes are typically conserved. To address this paradox, we have reconstructed computationally and resurrected in vivo the cis-regulatory regions of the ancestral Drosophila eve stripe 2 element and evaluated its evolution using a mathematical model of promoter function. Our feed-forward transcriptional model predicts gene expression patterns directly from enhancer sequence. We used this functional model along with phylogenetics to generate a set of possible ancestral eve stripe 2 sequences for the common ancestors of 1) D. simulans and D. sechellia; 2) D. melanogaster, D. simulans, and D. sechellia; and 3) D. erecta and D. yakuba. These ancestral sequences were synthesized and resurrected in vivo. Using a combination of quantitative and computational analysis, we find clear support for functional compensation between the binding sites for Bicoid, Giant, and Krüppel over the course of 40-60 My of Drosophila evolution. We show that this compensation is driven by a coupling interaction between Bicoid activation and repression at the anterior and posterior border necessary for proper placement of the anterior stripe 2 border. A multiplicity of mechanisms for binding site turnover exemplified by Bicoid, Giant, and Krüppel sites, explains how rapid sequence change may occur while maintaining the function of the cis-regulatory element.

Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene.

Here we present a quantitative and predictive model of the transcriptional readout of the proximal 1.7 kb of the control region of the Drosophila melanogaster gene even skipped (eve). The model is based on the positions and sequence of individual binding sites on the DNA and quantitative, time-resolved expression data at cellular resolution. These data demonstrated new expression features, first reported here. The model correctly predicts the expression patterns of mutations in trans, as well as point mutations, insertions and deletions in cis. It also shows that the nonclassical expression of stripe 7 driven by this fragment is activated by the protein Caudal (Cad), and repressed by the proteins Tailless (Tll) and Giant (Gt).

A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design.

Synthetic biology offers novel opportunities for elucidating transcriptional regulatory mechanisms and enhancer logic. Complex cis-regulatory sequences--like the ones driving expression of the Drosophila even-skipped gene--have proven difficult to design from existing knowledge, presumably due to the large number of protein-protein interactions needed to drive the correct expression patterns of genes in multicellular organisms. This work discusses two novel computational methods for the custom design of enhancers that employ a sophisticated, empirically validated transcriptional model, optimization algorithms, and synthetic biology. These synthetic elements have both utilitarian and academic value, including improving existing regulatory models as well as evolutionary questions. The first method involves the use of simulated annealing to explore the sequence space for synthetic enhancers whose expression output fit a given search criterion. The second method uses a novel optimization algorithm to find functionally accessible pathways between two enhancer sequences. These paths describe a set of mutations wherein the predicted expression pattern does not significantly vary at any point along the path. Both methods rely on a predictive mathematical framework that maps the enhancer sequence space to functional output.

Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants.

Massively parallel reporter assay measures transcriptional activities of various cis-regulatory modules (CRMs) in a single experiment. We developed a thermodynamic computational model framework that calculates quantitative levels of gene expression directly from regulatory DNA sequences. Using the framework, we investigated the molecular mechanisms of cis-regulatory mutations of a synthetic enhancer that cause abnormal gene expression. We found that, in a human cell line, competitive binding between family transcription factors (TFs) with slightly different binding preferences significantly increases the accuracy of recapitulating the transcriptional effects of thousands of single- or multi-mutations. We also discovered that even if various harmful mutations occurred in an activator binding site, CRM could stably maintain or even increase gene expression through a certain form of competitive binding between family TFs. These findings enhance understanding the effect of SNPs and indels on CRMs and would help building robust custom-designed CRMs for biologics production and gene therapy.

B-lines by lung ultrasound as a predictor of re-intubation in mechanically ventilated patients with heart failure.

Introduction: There have been few studies on predictors of weaning failure from MV in patients with heart failure (HF). We sought to investigate the predictive value of B-lines measured by lung ultrasound (LUS) on the risk of weaning failure from mechanical ventilation (MV) and in-hospital outcomes. Methods: This was a single-center, prospective observational study that included HF patients who were on invasive MV. LUS was performed immediate before ventilator weaning. A positive LUS exam was defined as the observation of two or more regions that had three or more count of B-lines located bilaterally on the thorax. The primary outcome was early MV weaning failure, defined as re-intubation within 72 h. Results: A total of 146 consecutive patients (mean age 70 years; 65.8% male) were enrolled. The total count of B-lines was a median of 10 and correlated with NT-pro-BNP level (r2 = 0.132, p < 0.001). Early weaning failure was significantly higher in the positive LUS group (9 out of 64, 14.1%) than the negative LUS group (2 out of 82, 2.4%) (p = 0.011). The rate of total re-intubation during the hospital stay (p = 0.004), duration of intensive care unit stay (p = 0.004), and hospital stay (p = 0.010) were greater in the positive LUS group. The negative predictive value (NPV) of positive LUS was 97.6% for the primary outcome. Conclusion: B-lines measured by LUS can predict the risk of weaning failure. Considering the high NPV of positive LUS, it may help guide the decision of weaning in patients on invasive MV due to acute decompensated HF.

Clinical implications of pleural effusion following left ventricular assist device implantation.

BACKGROUND: Studies on the association between pleural effusion (PE) and left ventricular assist devices (LVADs) are limited. This study aimed to examine the characteristics and the clinical impact of PE following LVAD implantation. METHODS: This study is a prospective analysis of patients who underwent LVAD implantation from June 2015 to December 2022. We investigated the prognostic impact of therapeutic drainage (TD) on clinical outcomes. We also compared the characteristics and clinical outcomes between early and late PE and examined the factors related to the development of late PE. RESULTS: A total of 71 patients was analyzed. The TD group (n=45) had a longer ward stay (days; median [interquartile range]: 31.0 [23.0-46.0] vs. 21.0 [16.0-34.0], P=0.006) and total hospital stay (47.0 [36.0-82.0] vs. 31.0 [22.0-48.0], P=0.002) compared to the no TD group (n=26). Early PE was mostly exudate, left-sided, and neutrophil-dominant even though predominance of lymphocytes was the most common finding in late PE. Patients with late PE had a higher rate of reintubation within 14 days (31.8% vs. 4.1%, P=0.004) and longer hospital stays than those without late PE (67.0 [43.0-104.0] vs. 36.0 [28.0-48.0], P<0.001). Subgroup analysis indicated that female sex, low body mass index, cardiac resynchronization therapy, and hypoalbuminemia were associated with late PE. CONCLUSIONS: Compared to patients not undergoing TD, those undergoing TD had a longer hospital stay but not a higher 90-day mortality. Patients with late PE had poor clinical outcomes. Therefore, the correction of risk factors, like hypoalbuminemia, may be required.

Identification of high sugar diet-induced dysregulated metabolic pathways in muscle using tissue-specific metabolic models in Drosophila.

Individual tissues perform highly specialized metabolic functions to maintain whole-body homeostasis. Although Drosophila serves as a powerful model for studying human metabolic diseases, a lack of tissue-specific metabolic models makes it challenging to quantitatively assess the metabolic processes of individual tissues and disease models in this organism. To address this issue, we reconstructed 32 tissue-specific genome-scale metabolic models (GEMs) using pseudo-bulk single cell transcriptomics data, revealing distinct metabolic network structures across tissues. Leveraging enzyme kinetics and flux analyses, we predicted tissue-dependent metabolic pathway activities, recapitulating known tissue functions and identifying tissue-specific metabolic signatures, as supported by metabolite profiling. Moreover, to demonstrate the utility of tissue-specific GEMs in a disease context, we examined the effect of a high sugar diet (HSD) on muscle metabolism. Together with 13C-glucose isotopic tracer studies, we identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a rate-limiting enzyme in response to HSD. Mechanistically, the decreased GAPDH activity was linked to elevated NADH/NAD+ ratio, caused by disturbed NAD+ regeneration rates, and oxidation of GAPDH. Furthermore, we introduced a pathway flux index to predict and validate additionally perturbed pathways, including fructose and butanoate metabolism. Altogether, our results represent a significant advance in generating quantitative tissue-specific GEMs and flux analyses in Drosophila, highlighting their use for identifying dysregulated metabolic pathways and their regulation in a human disease model.

Smartphone application-based rehabilitation in patients with chronic respiratory and cardiovascular diseases.

Rehabilitation improves symptoms, quality of life, and survival in patients with chronic respiratory or cardiovascular disease. We evaluated smartphone application-based rehabilitation programs for patients with chronic respiratory or cardiovascular diseases. This was a single-center prospective single arm study. Participants underwent smartphone application-based pulmonary or cardiac rehabilitation for 12 weeks. A total of 93 participants were recruited, and 75 visited after rehabilitation. Their median age was 67.0 (interquartile range, 60.0-70.8) years, and 60 (80.0%) were men. For patients with chronic respiratory disease (n = 41), VO2peak (median 13.7 to 15.4 ml/kg/min, P = 0.049), chronic obstructive pulmonary disease assessment test (median 14 to 6, P < 0.001), Euro-QoL 5-Dimension 5-Level (EQ-5D-5L) index (median 0.795 to 0.862, P = 0.001), and Health-related Quality of Life Instrument with 8 Items (HINT-8) index (median 0.784 to 0.855, P < 0.001) were significantly improved. For patients with chronic cardiovascular disease (n = 34), VO2peak (median 21.8 to 23.3, P = 0.007), EQ-5D-5L index (median 0.871 to 1.000, P = 0.037), and HINT-8 index (median 0.890 to 0.903, P < 0.001) were significantly improved. The smartphone application-based rehabilitation program improved exercise capacity and quality of life in patients with chronic respiratory or cardiovascular disease.Trial registration: https://clinicaltrials.gov/ct2/show/NCT05383950 (20/05/2022).

BMI1 regulates human erythroid self-renewal through both gene repression and gene activation.

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.

Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts.

Primitive erythroid cells, the first red blood cells produced in the mammalian embryo, are necessary for embryonic survival. Erythropoietin and its receptor EpoR, are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However, the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However, Epor-null embryos develop a progressive, profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression, from advanced cellular maturation, and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation, survival, and appropriate timing of terminal maturation of primitive erythroid precursors.