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

描述（由申请人提供）：造血系统起源于一小群自我更新的造血干细胞（hsc），而hsc又来源于成血管细胞，即血液和血管的常见前体。广泛的研究已经确定了关键的转录因子，如基本螺旋环螺旋（bLH）、SCL/TAL1和GATA因子，它们在造血干细胞向红系、髓系和淋巴系分化的连续步骤中发挥关键作用。尽管取得了这些进展，但使成血管细胞转化为血液和内皮细胞的因素的性质尚不清楚，在造血干细胞自我更新和谱系承诺中起作用的转录网络仍不完整。在初步研究中，我们发现kruppel样锌指转录因子ZBP-89是FLK1+中胚层早期向血液和血管发育的主要调控因子。强制表达ZBP-89增加了斑马鱼和小鼠胚胎干细胞/胚胎体（EB）培养物中原始和最终造血功能的表达，但减少了斑马鱼胚胎中轴向和体间血管的形成，以及小鼠EB培养物中内皮细胞的复制潜能和新生血管的生成。在斑马鱼或小鼠ESCs中，敲低ZBP-89导致原始和最终造血标志物（如SCL和GATA因子）的显著减少，但内皮谱系标志物的增加，将ZBP-89的作用机制与所有其他已知的调节血管发育的转录因子区分出来。此外，研究人员还发现，ZBP-89突变等位基因纯合的小鼠在围产期死亡，循环红细胞明显减少，但成熟髓系细胞增加，这反映了ZBP-89在胎儿造血中的另一个关键作用，可能是在双电位共同髓系祖（CMP）干细胞的水平上，这导致了红系和髓系血统的产生。ZBP-89调节对成人造血的影响及其在成血管细胞和cmp水平上参与的潜在转录网络尚不清楚。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（目的1），确定其异位表达对成年小鼠和斑马鱼血液和血管谱系发育的影响（目的2），并阐明ZBP-89介导的造血谱系承诺的机制。(3)为目标。将利用小鼠和斑马鱼的遗传、生化、蛋白质组学、基因组学、干细胞培养和骨髓移植。公共卫生相关性：所有血细胞的形成在发育过程中与血管的形成密切相关，这两个过程在很大程度上受转录因子的调节，转录因子是控制普通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。尽管这种决定细胞命运的指导网络对新疗法的发展有潜在的影响，但它仍然不明确。例如，血液干细胞被用于重建因放射或化疗而受损的骨髓，但它们的数量很少，这是细胞治疗的一个主要挑战。通过促进干细胞前体的发育来增加它们的数量将满足一个关键的需求。增强或抑制新血管形成（血管生成）的方法也可分别用于心脏病发作后的心脏修复或减少有害血管生成（由肿瘤或慢性炎症诱导）。我们已经发现了一种转录因子，它位于血液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展网络。

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.