Characterization and Molecular Regulation of Embryonic Hemogenic Endothelium

描述（由申请方提供）：造血是哺乳动物胚胎发育以及幼龄和成年动物生命维持的关键过程。在胚胎早期至中期发育期间的不良血液形成能力可导致产前死亡，而出生后生命中的缺陷或不受控制的血细胞分化和增殖可导致广泛的功能障碍和疾病，包括各种贫血、血液白血病/淋巴瘤和继发于B-和T-细胞谱系异常发育的免疫缺陷。目前存在两种关于导致早期造血细胞形成的祖细胞的理论;成血管细胞（一种产生造血干细胞（HSC）和生血内皮细胞（一种原始内皮细胞，其存在于发育中的脉管系统中并在血管腔内分化成造血细胞）的双能祖细胞）。我们实验室以前的工作已经确定了一群cKit+细胞，它们位于发育中的卵黄囊内的血管内皮内，当在MethoCult M3434培养基中培养时，通过cKit+ CD 45-细胞表面标志物表达进行分选时，产生造血集落。虽然在我们实验室进行的工作主要集中在卵黄囊上，因为它的分离简单，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，那里有明确的造血部位。提出的研究的目的是检验以下假设：即主动脉旁内脏胸膜/性腺中肾（PAS/AGM）区域内的Hoechst染料流出（SP）细胞是cKit+ Flk+，是能够产生所有血液谱系的生血内皮细胞，并且当移植到受辐射的动物受体中时具有再增殖能力。这一假说挑战了现有的所谓的成血管细胞作为发育中的哺乳动物胚胎中血液形成细胞的主要来源的范例。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，在那里造血被认为是发生，并确定其在体外的造血潜能。接下来，我们希望使用整体/冷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，并明确显示它们从内皮细胞分化为血管腔内的造血细胞。最后，我们打算表征这些细胞的细胞表型和分子调控，并通过qRT-PCR和微阵列分析确定重要/独特的基因表达谱。这项研究在公共卫生方面的应用在于，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.