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The transcription factor Myb is a key regulator of hematopoietic cell proliferation, differentiation, and survival and has been implicated in the development of leukemia and several other human cancers. Pharmacological inhibition of Myb is therefore emerging as a potential therapeutic strategy. Recently, the first low-molecular-weight compounds that show Myb inhibitory activity have been identified. Characterization of these compounds suggests disruption of the protein–protein–interaction of Myb and the coactivator p300 as a suitable strategy to inhibit Myb. [ABSTRACT FROM AUTHOR]

MicroRNAs are important regulators of normal and malignant hematopoiesis. A paper by Lu et al. in this issue of Developmental Cell demonstrates how one microRNA, miR-150, plays a critical role in commitment of the erythroid-megakaryocyte progenitor by modulating the level of the widely expressed transcription factor MYB (or c-Myb). [Copyright &y& Elsevier]

The cell-cycle-regulated Myb-family transcription factor B-Myb is crucial during S phase in many diploid cell types. We have examined the expression and function of B-Myb in megakaryocytic differentiation, during which cells progress from a diploid to a polyploid state. In contrast to terminal differentiation of most haematopoietic cells, during which B-myb is rapidly downregulated, differentiation of megakaryocytes is accompanied by continued B-myb RNA and protein expression. Overexpression of B-Myb in a megakaryoblastic cell line resulted in an increase in the number of cells entering S phase and, upon induction of differentiation, the fraction of cells actively endoreplicating increased. By contrast, reduction of B-Myb levels using short interfering (si)RNA resulted in a decline in S-phase progression during both normal and endoreplicative DNA synthesis. This effect correlated with aberrant localisation of initiation of DNA replication within the nucleus and an increased fraction of cells in mitosis. Chromosomal fragmentation and other aberrations, including shorter, thicker chromatids, end-to-end fusion, and loss of a chromatid, suggest that reduced B-Myb activity is also associated with structural chromosomal instability. [ABSTRACT FROM AUTHOR]

Objectives: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. Methods: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. Results: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. Conclusion: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells. [Copyright &y& Elsevier]

1 Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two Gprotein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to α[sub IIb]β[sub 3] integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. 2 In the presence of both fibrinogen and the α[sub IIb]β[sub 3] receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. 3 Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombininduced α[sub IIb]β[sub 3]-dependent aggregation and release, but had no effect on the secondary fibrindependent response. 4 Fixing of the platelets abolished α[sub Ib]β[sub 3]-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary 'aggregation'. 5 In conclusion, the secondary fibrin-dependent 'aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin. [ABSTRACT FROM AUTHOR]

Rearrangement of the FLI-1 locus with ensuing overexpression of ELI-1 is an early event in Friend murine leukemia virus-induced disease. When over-expressed in primary erythroblasts, ELI-1 blocks erythropoeitin (Epo)-induced terminal differentiation and inhibits apoptosis normally induced in response to Epo withdrawal. We show here that the survival-inducing property of ELI-i is associated with increased tran- scription of BCL-2. We further show that ELI-1 binds BCL-2 promoter sequences in transformed erythroblasts, and in vitro studies identify specific ELI-1-binding sites essential for the transactivation of the BCL-2 promoter by ELI-1. Analysis of ELI-1 mutants showed a correlation between the ability of ELI-1 to transactivate BCL-2 promoter sequences and their ability to inhibit apoptosis in the absence of Epo. Moreover, inhibitor studies confirmed the essential rote of BCL-2 for ELI-1-transformed erythroblast survival. Finally, enforced expression of BCL-2 was sufficient to pro- mote survival and terminal differentiation of erythroblasts in the absence of Epo. These results show that BCL-2 is an in vivo target of ELI-1 in ELI-1-transformed erythroblasts and that its deregulated expression is instrumental in the survival of these cells. [ABSTRACT FROM AUTHOR]

Cumulatively, breast, cervical, ovarian, and uterine cancer account for more than 70% of cancers in women in India. Distinct differences in the clinical presentation of women with cancer suggest underlying differences in cancer biology and genetics. The peak age of onset of breast and ovarian cancer appears to be a decade earlier in India (age 45-50 years) than in high-income countries (age >60 years). Understanding these differences through research to develop diagnosis, screening, prevention, and treatment frameworks that ar e specific to the Indian population are critical and essential to improving women's health in India. Since the sequencing of the human genome in 2001, applications of advanced technologies, such as massively parallel sequencing, have transformed the understanding of the genetic and environmental drivers of cancer. How can advanced technologies be harnessed to provide health-care solutions at a scale and to a budget suitable for a country of 1·2 billion people? What research programmes are necessary to answer questions specific to India, and to build capacity for innovative solutions using these technologies? In order to answer these questions, we convened a workshop with key stakeholders to address these issues. In this Series paper, we highlight challenges in tackling the growing cancer burden in India, discuss ongoing genomics research and developments in infrastructure, and suggest key priorities for future research in cancer in India. [ABSTRACT FROM AUTHOR]

Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activatorlike effector nucleases. FAH-/- rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wildtype rabbit primary hepatocytes into FAH-/- rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH-/- rabbits are an attractive alternative for modeling the consequences of HT1. [ABSTRACT FROM AUTHOR]

The transcription factor Myb plays a key role in the hematopoietic system and has been implicated in the development of leukemia and other human cancers. Inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. However, because of a lack of suitable inhibitors, the feasibility of therapeutic approaches based on Myb inhibition has not been explored. We have identified the triterpenoid Celastrol as a potent low-molecular-weight inhibitor of the interaction of Myb with its cooperation partner p300. We demonstrate that Celastrol suppresses the proliferative potential of acute myeloid leukemia (AML) cells while not affecting normal hematopoietic progenitor cells. Furthermore, Celastrol prolongs the survival of mice in a model of an aggressive AML. Overall, our work demonstrates the therapeutic potential of a small molecule inhibitor of the Myb/p300 interaction for the treatment ofAMLandprovides a starting point for the furtherdevelopment of Myb-inhibitorycompoundsfor the treatment of leukemia and, possibly, other tumors driven by deregulated Myb. [ABSTRACT FROM AUTHOR]

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 byactivation 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. [ABSTRACT FROM AUTHOR]

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor, CLEC-2, exhibit haemorrhaging 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 haemorrhages 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 haemorrhaging. Haemorrhaging 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 haemorrhage. [ABSTRACT FROM AUTHOR]

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activatorlike effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1,3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG 1/2 knockout pigs are promising tools for biomedical and translational research. [ABSTRACT FROM AUTHOR]

Objectives: Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment. Methods: Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1β or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally. Results: IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12. Conclusion: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential. [ABSTRACT FROM AUTHOR]

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact withADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8- ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting. [ABSTRACT FROM AUTHOR]

Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b locus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression. [ABSTRACT FROM AUTHOR]

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. CD11bhigh monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80bright 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Integrin αIIbβ3 is expressed in mast cells as well as in megakaryocytes/platelets. A recent study has shown that surface expression levels of integrin αVβ3 are elevated in integrin αIIb-deficient bone marrow-derived mast cells (BMMCs) as compared with wild-type (WT) counterparts, but the underlying mechanism remains obscure. Here we demonstrate by transducing integrin αllb into integrin αIIb-deficient BMMCs that surface expression levels of integrin αVβ3 are inversely related to those of integrin αIIbβ3. Thus, competitive association of integrin β3 with integrin αllb or integrin αV determines surface expression levels of integrin αIIbβ3 or αVβ3 in mast cells. We compared WT and integrin αlIb-deficient BMMCs as well as integrin αlIb-deficient BMMCs transduced with integrin αIIb(WT) or non-functional αlIb(D163A) mutant and found that enhancement of proliferation, degranulation, cytokine production, and migration of BMMCs through interaction with fibrinogen (FB) depended on integrin αIIbβ3. In addition, elevated surface expression of integrin αVβ3 failed to compensate for loss of FB-associated functions in integrin αIIb-deficient BMMCs while enhancing adhesion to vitronectin or von Willebrand factor. Importantly, integrin αlIb deficiency strongly suppressed chronic inflammation with the remarkable increase of mast cells induced by continuous intraperitoneal administration of FB, although it did not affect acute allergic responses or mast cell numbers in tissues in steady states. Interestingly, soluble FB promoted cytokine production of BMMCs in response to Staphylococcus aureus with FB-binding capacity, through integrin αIIbβ3-dependent recognition of this pathogen. Collectively, integrin αIIbβ3 in mast cells plays an important part in FB-associated, chronic inflammation and innate immune responses. [ABSTRACT FROM AUTHOR]

Murine haematopoietic stem cells (HSCs) are contained in the Kit+Sca1+Lin− (KSL) population of bone marrow and are able to repopulate lethally irradiated mice. Myeloproliferative disorders (MPDs) are thought to be clonogenic diseases arising at the level of the HSC. Here, we show that mice expressing low levels of the transcription factor c-Myb, as the result of genetic knockdown, develop a transplantable myeloproliferative phenotype that closely resembles the human disease essential thrombocythaemia (ET). Unlike wild-type cells, the KSL population in c-myb knockdown bone marrow cannot repopulate irradiated mice and does not transfer the disease. Instead, cells positive for Kit and expressing low to medium levels of CD11b acquire self-renewing stem cell properties and are responsible for the perpetuation of the myeloproliferative phenotype. [ABSTRACT FROM AUTHOR]

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1-/- mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1-/- bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood [ABSTRACT FROM AUTHOR]

The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation. [ABSTRACT FROM AUTHOR]

Objective: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis. Methods: A two-step serum-free method was developed. Cells isolated using this method were analyzed for surface marker expression by flow cytometry, and for their ability to differentiate using single-cell culture. Purified progenitors were induced to differentiate and analyzed with respect to their ploidy by flow cytometry and expression of specific genes by RT-PCR. Results: A population of Lin−c-kit+CD45+CD41+CD31+CD34lowCD9lowFcγRII/IIIlowSca-1med/low committed megakaryocyte progenitors was purified. These cells could be differentiated efficiently, achieving ploidy of up to 128N. Analysis of RNA demonstrated the expected increases in expression of key megakaryocyte-associated genes. RT-PCR analysis also revealed that a range of Ets and GATA factors are expressed, their individual levels and patterns of expression varying widely. Surprisingly, we find that GATA-6 is specifically expressed in late differentiated megakaryocytes and has the potential to regulate megakaryocyte-expressed genes in cooperation with Ets factors. Conclusion: Purified primary megakaryocytic progenitors are able to differentiate as a cohort into fully mature megakaryocytes. The number of cells obtainable, and the synchrony of the differentiation process, facilitates analysis of the dynamics of molecular processes involved in megakaryocytopoiesis. The expression pattern of Ets and GATA family transcription factors reveals the complexity of the involvement of these key megakaryocytic regulators. The finding of GATA-6 expression and demonstration of its functional activity suggests a novel mechanism for the regulation of certain genes late in megakaryocytopoiesis. [Copyright &y& Elsevier]

Previous studies have identified an enhancer 3' of the scl gene that can direct transgene expression to hematopoietic progenitors and stem cells. Here we use this enhancer to restrict expression of the avian leukosis virus receptor, TVA, to hematopoietic stem cells and progenitors in bone marrow and fetal liver and demonstrate that retroviral infection can be used to specifically introduce exogenous sequences. We show that a majority of CFU-S[sub 12] multipotential progenitor cells can be transduced in vitro. Uniquely, transduction of TVA[sup +] progenitors with a retrovirus encoding a puromycin resistance gene allows selection and expansion of a multipotential hematopoietic progenitor population that can be superinfected with high efficiency. Using this system we show for the first time that v-Myb oncoproteins expressed from avian viruses can induce a leukemic transformation in the mouse. The phenotype of the transformed cells is similar to that which is seen in the chicken and is likewise dependent on the particular structure of v-Myb. This implies that the basic mechanisms of action of mutated transcription factors in the etiology of leukemia are conserved between birds and mammals. [ABSTRACT FROM AUTHOR]

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 α[sub 2]β[sub 1] and activation through the glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain complex. The present study extends this model by demonstrating that engagement of α[sub 2]β[sub 1] 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, α[sub 2]β[sub 1] 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 PLCγ,2 as well as plasma membrane calcium ATPase and focal adhesion kinase, α[sub 2]β[sub 1]-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 Ca[sup 2+]. Demonstration that adhesion of platelets to collagen via α[sub 2]β[sub 1] generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of β[sub 1]-deficient thrombi and why loss of the GPVI-FcR γ-chain complex has a relatively minor effect on bleeding. [ABSTRACT FROM AUTHOR]

Characterizes the c-Myb-responsive region and regulation of the human type I collagen alpha2 chain gene by c-Myb. Identification of four Myb-binding sites in the promoter; Transactivation assays on wild type and mutant promoter-reporter constructs; Expression of fibronectin and type III collagen; Mechanisms in the pathogenesis of several fibrotic disorders.

This review summarises some of the key developments that have taken place in our understanding of platelet-collagen interactions within the last 18 months. Within this time, the major activatory collagen receptor glycoprotein VI (GPVI) has been sequenced and shown to reconstitute collagen responses in a megakaryocytic cell line. It is a member of the Ig superfamily of proteins, with two extracellular Ig domains, and is constitutively associated with the Fc receptor γ-chain (FcR γ-chain). GPVI signals through a pathway that shares many features with those of immune receptors, with critical roles for Syk and the adapters LAT and SLP-76 in the activation of PLCγ2. Significant developments have also taken place in regard to the role of the major adhesion receptor for collagen, the integrin α2β1 (also known as GPIa-IIa). An α2β1-selective collagen-based peptide has been developed and co-crystalisalised with the I-domain of the α2 subunit. Polymorphisms in α2 have been shown to cause wide variation in expression of α2β1, with the α2 allele T[sub 807]/A[sub 873] leading to a high level of the integrin and increased risk of stroke in young people. Activation of platelets by a wide range of agonists has been shown to increase the affinity of α2β1 to intermediate or high affinity states. This has important implications for the two-site, two-state model of collagen-platelet interactions. A new model is proposed in which collagen binds initially to either α2β1 or GPVI, leading to subsequent binding to the other receptor and conversion of the integrin to a high affinity state. In this model, both receptors generate intracellular signals which support platelet activation. [ABSTRACT FROM AUTHOR]