Characterization and Molecular Regulation of Embryonic Hemogenic Endothelium

描述(申请人提供)：无论是哺乳动物胚胎的发育，还是幼年和成年动物的生命维持，造血都是一个关键的过程。胚胎发育早期和中期的造血能力缺陷可能会导致出生前死亡，而出生后生命中缺乏或不受控制的血细胞分化和增殖可能会导致广泛的功能障碍和疾病，包括各种贫血、血液白血病/淋巴瘤，以及继发于B和T细胞谱系异常发育的免疫缺陷。目前关于导致早期造血细胞形成的前体细胞有两种理论：血管母细胞(一种同时产生造血干细胞(HSC)的双功能前体细胞)和血源性内皮(一种原始内皮细胞，位于发育中的血管系统中，并在血管腔内分化为造血细胞)。我们实验室以前的工作已经定义了一组cKit+细胞，这些细胞驻留在发育中的卵黄囊内的血管内皮细胞中，当按cKit+CD45-细胞表面标记进行分类时，在MethoCult M3434培养液中培养时会产生造血细胞集落。虽然我们实验室的工作主要集中在卵黄囊上，因为它分离简单，细胞类型相对较少，但我们现在渴望在胚胎中测试我们的发现，那里是确定的造血部位。这项研究的目的是验证一种假设，即主动脉旁内脏胸膜/主动脉-性腺-中肾(PAS/AGM)区域内cKit+Flk+的Hoechst染料流出(SP)细胞是能够产生所有血系的造血内皮细胞，当移植到受照射的动物体内时，具有再繁殖能力。这一假说挑战了现有的所谓血管母细胞作为发育中的哺乳动物胚胎中主要造血细胞来源的范式。为了验证这一假设，我们的第一个目标是从PAS/AGM区域分离SP细胞，并在体外确定它们的最终造血潜力。下一步，我们希望使用整体贴片/冰冻切片染色以及体内成像来跟踪这些细胞的整个发育过程，并明确显示它们从驻留在内皮细胞到血管腔内的造血细胞的分化。最后，我们打算通过qRT-PCR和微阵列分析来表征这些细胞的细胞表型和分子调控，并确定重要/独特的基因表达谱。这项研究对公共卫生的应用在于，了解血管和细胞的发育将有助于人类疾病的治疗方法。

DESCRIPTION (provided by applicant): Hematopoiesis is a critical process in both the development of mammalian embryos and sustaining of life in young and adult animals. Faulty blood-forming capabilities during early-to mid-development of an embryo can lead to pre-natal death, while deficiencies or uncontrolled blood cell differentiation and proliferation in postnatal life can lead to a wide range of dysfunction and disease including various anemias, blood leukemias/lymphomas, and immune deficiencies secondary to aberrant development of the B- and T- cell lineages. There currently exist two theories regarding the progenitor cell that leads to formation of early hematopoietic cells; the hemangioblast (a bi-potent progenitor cell that gives rise to both hematopoietic stem cells (HSC) and the hemogenic endothelium (a primitive endothelial cell that resides in the developing vasculature and differentiates into blood-forming cells within the vessel lumen). Previous work in our lab has defined a population of cKit+ cells that reside within the vascular endothelium within the developing yolk sac, and when sorted by cKit+ CD45- cell surface marker expression give rise to hematopoietic colonies when cultured in MethoCult M3434 media. While the work conducted in our lab has focused primarily on the yolk sac due to its simplicity of isolation and relative paucity of cell types, we are now eager to test our findings in the embryo proper, where sites of definitive hematopoiesis reside. The goal of the research proposed is to test the hypothesis that Hoechst dye-effluxing (SP) cells that are cKit+ Flk+ within the para-aortic splanchnopleure/aorta-gonad-mesonephros (PAS/AGM) region are hemogenic endothelial cells capable of giving rise to all blood lineages and have repopulation capabilities when transplanted into irradiated animal recipients. This hypothesis challenges the existing paradigm of the so-called hemangioblast as the predominant source of blood forming cells in the developing mammalian embryo. In order to test this hypothesis, our first aim is to isolate the SP cells from the PAS/AGM region, where definitive hematopoiesis is thought to occur, and determine their definitive hematopoietic potential in vitro. Next, we would like to use both whole mount/frozen section staining as well as in vivo imaging to track these cells throughout development and definitively show their differentiation from cells residing in the endothelium to blood-forming cells within the vessel lumen. Finally, we intend to characterize the cellular phenotype and molecular regulation of these cells and determine important/unique gene expression profiles by qRT-PCR and microarray analysis. The application of this research for public health lies in the fact that understanding the development of blood vessels and cells will aid in therapeutic approaches for human disease.