Role of the zinc finger transcription factor ZBP89 in blood & vascular developmen

描述(申请人提供)：造血系统起源于一小群自我更新的造血干细胞(HSCs)，而HSCs又来自血管母细胞，血管和血管的共同前体。广泛的研究已经确定了关键的转录因子，如碱性螺旋环螺旋(BLH)SCL/TAL1和GATA因子，它们在HSC向红系、髓系和淋巴系分化的后续步骤中发挥关键作用。尽管取得了这些进展，但将血管母细胞与血液和内皮细胞联系起来的因素的性质尚不清楚，参与HSC自我更新和谱系承诺的转录网络仍然不完整。在初步研究中，我们已经确定Kruppel类锌指转录因子ZBP-89是Flk1+中胚层发育成血管的主要调节因子。ZBP-基因的强制表达增加了斑马鱼和小鼠胚胎干细胞(ESCs)/胚体(EB)培养中原始和确定的造血细胞的表达，但降低了斑马鱼胚胎中轴向和体间血管的形成以及小鼠EB培养中的内皮复制能力和萌芽血管生成。ZBP-89在斑马鱼或小鼠胚胎干细胞中的敲除导致原始和决定性的造血标记物(如SCL和GATA因子)显著减少，但内皮细胞系标记物增加，从而使ZBP-89的作用机制有别于所有其他已知的调节血管发育的转录因子。此外，我们发现具有低态ZBP-89突变等位基因纯合的小鼠在围产期死亡，循环中的红细胞显著减少，但成熟的髓系细胞增加，反映了ZBP-89在胎儿造血中的额外关键作用，可能在双潜能共同髓系祖细胞(CMP)干细胞的水平上，从而产生红系和髓系。ZBP-89对成人造血的调节作用以及在血管母细胞和CMPS水平上参与其作用的潜在转录网络尚未被探索。在这项应用中，我们建议评估ZBP-89缺失对胎儿和成人体内造血和血管发育的影响(目标1)，确定其异位表达对成年小鼠和斑马鱼血管和血管谱系发育的影响(目标2)，并阐明ZBP-89介导的造血谱系承诺的机制(S)。(目标3)。将利用遗传学、生化、蛋白质组学、基因组学、干细胞培养和小鼠和斑马鱼的骨髓移植。公共卫生相关性：所有血细胞的形成在发育上与血管的形成密切相关，这两个过程在很大程度上受到转录因子的调节，转录因子是控制常见的未分化干细胞前体成为血细胞或血管细胞的决定的蛋白质。作为细胞命运决定基础的指导网络仍然不明确，尽管它对新疗法的开发有潜在的影响。例如，血液干细胞正被用来重建因放射或化疗而受损的骨髓，但它们的数量非常少，这是细胞治疗中的一大挑战。通过促进干细胞前体细胞的发育来增加它们的数量将满足迫切的需求。增强或抑制新血管形成(血管生成)的方法也可能分别用于心脏病发作后的心脏修复或减少有害的血管生成(由肿瘤或慢性炎症引起)。我们发现了一种转录因子，它位于血管和血管发育的十字路口。我们建议进行一系列研究，以确定这一因素的作用，并确定它所协调的发育网络。

DESCRIPTION (provided by applicant): The hematopoietic system originates from a small population of self-renewing hematopoietic stem cells (HSCs) that in turn derive from hemangioblasts, common precursors of blood and blood vessels. Extensive studies have identified key transcription factors, such as the basic helix loop helix (bLH) SCL/TAL1 and GATA factors, which play critical roles in the successive steps of differentiation of HSCs into the erythroid, myeloid and lymphoid lineages. Despite these advances, the nature of the factors that commit the hemangioblast to blood and endothelial cells are obscure and the transcriptional networks operative in HSC self-renewal and lineage commitment remain incomplete. In preliminary studies, we have identified the Kruppel-like zinc finger transcription factor ZBP-89 as a master regulator of early development of FLK1+ mesoderm into blood and blood vessels. Forced expression of ZBP-89 increased expression of primitive and definitive hematopoiesis in zebrafish and in mouse embryonic stem cells (ESCs)/embryonic body (EB) cultures, but reduced axial and intersomitic blood vessel formation in zebrafish embryos and the endothelial replating potential and sprouting angiogenesis in mouse EB cultures. Knockdown of ZBP-89 in zebrafish or in mouse ESCs resulted in a dramatic reduction in primitive and definitive hematopoietic markers (e.g. SCL and GATA factors), but an increase in endothelial lineage markers, distinguishing the mechanism of action of ZBP-89 from all other known transcription factors regulating hematovascular development. Further, we find that mouse pubs homozygous for a hypomorphic ZBP-89 mutant allele die perinatally and exhibit a marked reduction in circulating red blood cells, but an increase in mature myeloid cells, reflecting an additional critical role for ZBP-89 in fetal hematopoiesis, perhaps at the level of the bipotential Common Myeloid Progenitor (CMP) stem cell, which gives rise to both the erythroid and myeloid lineages. The effects of ZBP-89 modulation on adult hematopoiesis and the underlying transcriptional networks involved in its action at the level of hemangioblasts and CMPs are unexplored. In this application, we propose to assess the consequences of loss of ZBP-89 on fetal and adult hematopoiesis and vascular development in vivo (Aim 1), determine the effects of its ectopic expression on blood and vessel lineage development in adult mice and zebrafish (Aim 2), and elucidate the mechanism(s) underlying ZBP-89- mediated hematopoietic lineage commitment. (Aim 3). Genetic, biochemical, proteomics, genomics, stem cell cultures and bone marrow transplantation in mouse and zebrafish will be utilized. PUBLIC HEALTH RELEVANCE: Formation of all blood cells is closely linked developmentally to formation of blood vessels and the two processes are regulated in large part by transcription factors, proteins that control the decision of a common undifferentiated stem cell precursor to become a blood cell or a vascular cell. The instructional network that underlies this cell fate decision remains ill defined, despite its potential impact on development of new therapies. For example, blood stem cells are being used to reconstitute bone marrow damaged by radiation or chemotherapy, but they are very few in numbers, a major challenge in cellular therapy. Increasing their number by enhancing their development from their stem cell precursors will serve a critical need. Methods for enhancing or suppressing new blood vessel formation (angiogenesis) may also be useful respectively, in cardiac repair following a heart attack or in reducing harmful angiogenesis (induced by tumors or chronic inflammation). We have discovered a transcription factor that lies at the cross road of blood and blood vessel development. We are proposing a series of studies to pinpoint the role of this factor, and define the developmental networks it coordinates.