The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors.

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice.

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

dropClust: efficient clustering of ultra-large scRNA-seq data.

Droplet based single cell transcriptomics has recently enabled parallel screening of tens of thousands of single cells. Clustering methods that scale for such high dimensional data without compromising accuracy are scarce. We exploit Locality Sensitive Hashing, an approximate nearest neighbour search technique to develop a de novo clustering algorithm for large-scale single cell data. On a number of real datasets, dropClust outperformed the existing best practice methods in terms of execution time, clustering accuracy and detectability of minor cell sub-types.

The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

Identification of unipotent megakaryocyte progenitors in human hematopoiesis.

The developmental pathway for human megakaryocytes remains unclear, and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem- and progenitor-cell populations that specifically expresses genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34+CD38+IL-3RαdimCD45RA- common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte-macrophage potential but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro. The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41+ CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.

Identifying and enriching platelet-producing human stem cell-derived megakaryocytes using factor V uptake.

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

CD45 expression discriminates waves of embryonic megakaryocytes in the mouse.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45- cells. These populations, and that of CD41++CD45-CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45-CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41-CD45++CD11b+ cells, which produce low numbers of CD41++CD45-CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45-CD42c+ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45-CD42c+ embryo megakaryocytes that can be produced from CD41+CD45- or from CD41+CD45+ cells, at difference from those from bone marrow.

Cytokine control of megakaryopoiesis.

Platelets are anuclear blood cells required for haemostasis and are implicated in other processes including inflammation and metastasis. Platelets are produced by megakaryocytes, specialized cells that are themselves generated by a process of controlled differentiation and maturation of bone-marrow stem and progenitor cells. This process of megakaryopoiesis involves the coordinated interplay of transcription factor-controlled cellular programming with extra-cellular cues produced locally in supporting niches or as circulating factors. This review focuses on these external cues, the cytokines and chemokines, that drive production of megakaryocytes and support the terminal process of platelet release. Emphasis is given to thrombopoietin (Tpo), the major cytokine regulator of steady-state megakaryopoiesis, and its specific cell surface receptor, the Mpl protein, including normal and pathological roles as well as clinical application. The potential for alternative or supplementary regulatory mechanisms for platelet production, particularly in times of acute need, or in states of infection or inflammation are also discussed.

Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets.

The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production.

miR-125b-2 is a potential oncomiR on human chromosome 21 in megakaryoblastic leukemia.

Children with trisomy 21/Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the related transient leukemia (DS-TL). The factors on human chromosome 21 (Hsa21) that confer this predisposing effect, especially in synergy with consistently mutated transcription factor GATA1 (GATA1s), remain poorly understood. Here, we investigated the role of Hsa21-encoded miR-125b-2, a microRNA (miRNA) overexpressed in DS-AMKL/TL, in hematopoiesis and leukemogenesis. We identified a function of miR-125b-2 in increasing proliferation and self-renewal of human and mouse megakaryocytic progenitors (MPs) and megakaryocytic/erythroid progenitors (MEPs). miR-125b-2 overexpression did not affect megakaryocytic and erythroid differentiation, but severely perturbed myeloid differentiation. The proproliferative effect of miR-125b-2 on MEPs accentuated the Gata1s mutation, whereas growth of DS-AMKL/TL cells was impaired upon miR-125b repression, suggesting synergism during leukemic transformation in GATA1s-mutated DS-AMKL/TL. Integrative transcriptome analysis of hematopoietic cells upon modulation of miR-125b expression levels uncovered a set of miR-125b target genes, including DICER1 and ST18 as direct targets. Gene Set Enrichment Analysis revealed that this target gene set is down-regulated in DS-AMKL patients highly expressing miR-125b. Thus, we propose miR-125b-2 as a positive regulator of megakaryopoiesis and an oncomiR involved in the pathogenesis of trisomy 21-associated megakaryoblastic leukemia.

Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis.

Oncogene-mediated transformation of hematopoietic cells has been studied extensively, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay of signaling pathways and oncogenes is essential for developing targeted cancer therapies. Here, we address the vexing issue of how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by the triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21. We demonstrate overactivity of insulin-like growth factor (IGF) signaling in authentic human DS-AMKL and in a DS-AMKL mouse model generated through retroviral insertional mutagenesis. Fetal but not adult megakaryocytic progenitors are dependent on this pathway. GATA1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. Failure of a direct GATA1-E2F interaction in mutated GATA1s converges with overactive IGF signaling to promote cellular transformation of DS fetal progenitors, revealing a complex, fetal stage-specific regulatory network. Our study underscores context-dependent requirements during oncogenesis, and explains resistance to transformation of ostensibly similar adult progenitors.

Regulated expression of the prostacyclin receptor (IP) gene by androgens within the vasculature: Combined role for androgens and serum cholesterol.

The prostanoid prostacyclin plays a key cardioprotective role within the vasculature. There is increasing evidence that androgens may also confer cardioprotection but through unknown mechanisms. This study investigated whether the androgen dihydrotestosterone (DHT) may regulate expression of the prostacyclin/I prostanoid receptor or, in short, the IP in platelet-progenitor megakaryoblastic and vascular endothelial cells. DHT significantly increased IP mRNA and protein expression, IP-induced cAMP generation and promoter (PrmIP)-directed gene expression in all cell types examined. The androgen-responsive region was localised to a cis-acting androgen response element (ARE), which lies in close proximity to a functional sterol response element (SRE) within the core promoter. In normal serum conditions, DHT increased IP expression through classic androgen receptor (AR) binding to the functional ARE within the PrmIP. However, under conditions of low-cholesterol, DHT led to further increases in IP expression through an indirect mechanism involving AR-dependent upregulation of SCAP expression and enhanced SREBP1 processing & binding to the SRE within the PrmIP. Chromatin immunoprecipitation assays confirmed DHT-induced AR binding to the ARE in vivo in cells cultured in normal serum while, in conditions of low cholesterol, DHT led to increased AR and SREBP1 binding to the functional ARE and SRE cis-acting elements, respectively, within the core PrmIP resulting in further increases in IP expression. Collectively, these data establish that the human IP gene is under the transcriptional regulation of DHT, where this regulation is further influenced by serum-cholesterol levels. This may explain, in part, some of the protective actions of androgens within the vasculature.

RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation.

The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34(+) progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program. Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program.

Horizontal RNA transfer mediates platelet-induced hepatocyte proliferation.

Liver regeneration is stimulated by blood platelets, but the molecular mechanisms involved are largely unexplored. Although platelets are anucleate, they do contain coding or regulatory RNAs that can be functional within the platelet or, after transfer, in other cell types. Here, we show that platelets and platelet-like particles (PLPs) derived from the megakaryoblastic cell line MEG-01 stimulate proliferation of HepG2 cells. Platelets or PLPs were internalized within 1 hour by HepG2 cells and accumulated in the perinuclear region of the hepatocyte. Platelet internalization also occurred following a partial hepatectomy in mice. Annexin A5 blocked platelet internalization and HepG2 proliferation. We labeled total RNA of MEG-01 cells by incorporation of 5-ethynyluridine (EU) and added EU-labeled PLPs to HepG2 cells. PLP-derived RNA was detected in the cytoplasm of the HepG2 cell. We next generated PLPs containing green fluorescent protein (GFP)-tagged actin messenger RNA. PLPs did not synthesize GFP, but in coculture with HepG2 cells, significant GFP protein synthesis was demonstrated. RNA-degrading enzymes partly blocked the stimulating effect of platelets on hepatocyte proliferation. Thus, platelets stimulate hepatocyte proliferation via a mechanism that is dependent on platelet internalization by hepatocytes followed by functional transfer of RNA stored in the anucleate platelet. This mechanism may contribute to platelet-mediated liver regeneration.

Dual role of IL-21 in megakaryopoiesis and platelet homeostasis.

Gene profiling studies have indicated that in vitro differentiated human megakaryocytes express the receptor for IL-21 (IL-21R), an immunostimulatory cytokine associated with inflammatory disorders and currently under evaluation in cancer therapy. The aim of this study was to investigate whether IL-21 modulates megakaryopoiesis. We first checked the expression of IL-21 receptor on human bone marrow and in vitro differentiated megakaryocytes. We then investigated the effect of IL-21 on the in vitro differentiation of human blood CD34+ progenitors into megakaryocytes. Finally, we analyzed the consequences of hydrodynamic transfection-mediated transient expression of IL-21, on megakaryopoiesis and thrombopoiesis in mice. The IL-21Rα chain was expressed in human bone marrow megakaryocytes and was progressively induced during in vitro differentiation of human peripheral CD34+ progenitors, while the signal transducing γ chain was down-regulated. Consistently, the STAT3 phosphorylation induced by IL-21 diminished during the later stages of megakaryocytic differentiation. In vitro, IL-21 increased the number of colony-forming unit megakaryocytes generated from CD34+ cells and the number of megakaryocytes differentiated from CD34+ progenitors in a JAK3- and STAT3-dependent manner. Forced expression of IL-21 in mice increased the density of bi-potent megakaryocyte progenitors and bone marrow megakaryocytes, and the platelet generation, but increased platelet clearance with a consequent reduction in blood cell counts. Our work suggests that IL-21 regulates megakaryocyte development and platelet homeostasis. Thus, IL-21 may link immune responses to physiological or pathological platelet-dependent processes.

Effects of metformin on proliferation and apoptosis of human megakaryoblastic Dami and MEG-01 cells.

Metformin has received increasing attention for its potential anticancer activity against certain human leukemia cells, but its effects on human megakaryoblastic cells are unclear. This study aimed to investigate the effects of metformin on proliferation and apoptosis of human megakaryoblastic cells (Dami and MEG-01) and the underlying molecular mechanisms. CCK8 assay was employed to measure cell proliferation. Flow cytometry was adopted to detect cell apoptosis. Western blot was further employed to measure apoptosis-related proteins. In Dami and MEG-01 cells, metformin significantly inhibited proliferation and promoted apoptosis in a dose- and time-dependent manner, and metformin (4 mM) was selected for subsequent experiments. Metformin inhibited ERK1/2, JNK, and PI3K/Akt, but activated p38 pathway in these two cells. Moreover, inhibition of ERK1/2, JNK or PI3K/Akt pathway alone induced cell apoptosis compared to the control group. The combination of specific inhibitors of ERK1/2, JNK or PI3K/Akt pathway and metformin further promoted cell apoptosis and the up-regulation of p21, Bax, Bad, cleaved caspase-3 and -9 as well as the down-regulation of Bcl-2 mediated by metformin alone, but inhibition of p38 pathway exhibited the opposite results. These findings support the possibility of metformin treatment as a new therapeutic strategy against acute megakaryoblastic leukemia (AMKL).

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors.

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.

A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo.

In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.