Macrophages and dendritic cells (DCs) are key components of cellular immunity and are thought to originate and renew from hematopoietic stem cells (HSCs). However, some macrophages develop in the ...embryo before the appearance of definitive HSCs. We thus reinvestigated macrophage development. We found that the transcription factor Myb was required for development of HSCs and all CD11b high monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80 bright macrophages in several tissues, such as liver Kupffer cells, epidermal Langerhans cells, and microglia— cell populations that all can persist in adult mice independently of HSCs. These results define a lineage of tissue macrophages that derive from the YS and are genetically distinct from HSC progeny.
Recent studies have boosted our understanding of long noncoding RNAs （IncRNAs） in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression ...profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 （lincRNAop21） impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.
The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus ...formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.
•Fibrin is a novel ligand for the platelet collagen receptor, GPVI, increasing platelet procoagulant activity.•Activation of GPVI by fibrin contributes to thrombus growth and stabilization.
The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin⁺ CD45⁺ (VE-cad⁺CD45⁺) population ...contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study, we developed a new coaggregation culture system, which supports maturation of a novel population of CD45-negative (VE-cad⁺CD45⁻CD41⁺) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad⁺CD45⁺ pre-HSCs (termed here type I and type II pre-HSCs, respectively), thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta, we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers, in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs, in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal.
Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an ...endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP(+) HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.
Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this ...defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplaninfl/fl mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.
•Podoplanin and CLEC-2 critically drive the formation and integrity of developing cerebral blood vessels.•Loss of cerebrovascular integrity is influenced by the loss of αIIb-mediated platelet aggregation and platelet secretion.
The ductus arteriosus (DA) is a fetal shunt vessel between the pulmonary artery and the aorta that closes promptly after birth. Failure of postnatal DA closure is a major cause of morbidity and ...mortality particularly in preterm neonates. The events leading to DA closure are incompletely understood. Here we show that platelets have an essential role in DA closure. Using intravital microscopy of neonatal mice, we observed that platelets are recruited to the luminal aspect of the DA during closure. DA closure is impaired in neonates with malfunctioning platelet adhesion or aggregation or with defective platelet biogenesis. Defective DA closure resulted in a left-to-right shunt with increased pulmonary perfusion, pulmonary vascular remodeling and right ventricular hypertrophy. Our findings indicate that platelets are crucial for DA closure by promoting thrombotic sealing of the constricted DA and by supporting luminal remodeling. A retrospective clinical study revealed that thrombocytopenia is an independent predictor for failure of DA closure in preterm human newborns, indicating that platelets are likely to contribute to DA closure in humans.
Mesenchymal stem cells (MSC) have immunomodulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow-derived MSC with ...EC and found that MSC could downregulate adhesion of flowing neutrophils or lymphocytes and their subsequent transendothelial migration. This applied for EC treated with tumor necrosis factor-α (TNF), interleukin-1β (IL-1), or TNF and interferon-γ combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL-6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralizing antibody against IL-6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL-6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knockdown of IL-6-receptor expression in MSC as well as EC inhibited anti-inflammatory effects. This was explained when we detected soluble IL-6R receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralization of transforming growth factor-β indicated that activation of this factor in coculture contributed to IL-6 production. Thus, crosstalk between MSC and EC caused upregulation of production of IL-6 by MSC which in turn downregulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL-6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease.
The involvement of the transcription factor c-Myb in promoting the proliferation and inhibition of erythroid cell differentiation has been established in leukemia cell models. The anemia phenotype ...observed in c-myb knockout and knockdown mice highlights a critical role for c-Myb in erythropoiesis. However, determining the reason for the failure of erythropoiesis in these mice and the precise function of c-Myb in erythroid progenitors remains elusive. We examined erythroid development under conditions of reduced c-Myb protein levels and report an unexpected role for c-Myb in the promotion of commitment to the erythroid lineage and progression to erythroblast stages. c-myb knockdown erythroid colony-forming unit (CFU-E) stage progenitors displayed an immature phenotype and aberrant expression of several hematopoietic regulators. To extend our findings, we analyzed the response of normal enriched erythroid progenitors to inducible disruption of a floxed c-myb allele. In agreement with the c-myb knockdown phenotype, we show that c-Myb is strictly required for expression of the c-Kit receptor in erythroid cells.
Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet–collagen interaction, adhesion is mediated ...via the inside-out regulation of integrin α2β1 and activation through the glycoprotein VI (GPVI)–Fc receptor (FcR) γ-chain complex. The present study extends this model by demonstrating that engagement of α2β1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase–based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI–FcR γ-chain complex. The same events do not occur in platelet suspensions. α2β1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI–FcR γ-chain cascade, including Src, Syk, SLP-76, and PLCγ2 as well as plasma membrane calcium ATPase and focal adhesion kinase. α2β1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCγ2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via α2β1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of β1-deficient thrombi and why loss of the GPVI–FcR γ-chain complex has a relatively minor effect on bleeding.
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