Hematopoietic Commitment: Molecular Mechanisms

描述（由申请人提供）：我们的长期目标是了解造血系统是如何在发育中的胚胎中建立的。我们对体外分化胚胎干细胞（ES）的研究表明，造血系统是通过不同的、顺序产生的Flk-1和scl表达细胞建立的。Flk-I+SCL -细胞首先出现在发育中的胚状体（EBs，胚胎干细胞的体外分化后代）。Scl基因在Flk-1 + Scl -细胞内开启，产生Flk-1 + Scl +细胞。成血管细胞群是造血细胞和内皮细胞的共同祖细胞，在这些细胞中富集。在Flk-I+SCL +细胞中，Flk-1被下调，最终产生Flk-I-SCL +造血祖细胞。体外无血清胚胎干细胞分化模型发现，骨形态发生蛋白(BMP) -4在Flk-1 +和SCL +细胞的生成中起关键作用。许多研究暗示BMP-4在造血发育中的重要性。然而，没有一项研究调查了BMP-4发挥作用的确切发育阶段。我们的建议是进一步表征控制Flk-1和Scl表达的分子机制，并确定BMP-4建立造血系统的确切发育阶段。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the hematopoietic system is established in the developing embryo. Our studies of in vitro differentiated embryonic stem (ES) cells indicate that the hematopoietic system is established via distinct, sequentially generated Flk-1 and SCL-expressing cells. Flk-I+SCL - cells first arise in developing embryoid bodies (EBs, in vitro differentiated progeny of ES cells). The Scl gene is turned on within Flk-1 + SCL- cells to give rise to Flk-1 + SCL + cells. The hemangioblast cell population, a common progenitor of hematopoietic and endothelial cells, was enriched within these cells. Within Flk-I+SCL + cells, Flk-1 is down regulated to finally generate Flk-I-SCL + hematopoietic progenitors. A serum free in vitro differentiation model of ES cells has identified that bone morphogenetic protein (BMP) -4 is critical in the generation of Flk-1 + and SCL + cells. Many studies implicate the importance of BMP-4 in hematopoietic development. However, none of the studies examined the precise developmental stage in which the BMP-4 functions. Our proposal is to further characterize molecular mechanisms controlling Flk-1 and Scl expression and to determine the precise developmental stage where BMP-4 functions to establish the hematopoietic system. Specific aim I: Further characterize molecular events involved in the generation of Flk-1 + and SCL + cells. Our studies indicate that the activation of both map kinase and Smadl pathways by BMP-4 is critical for the generation of Flk-1 + cells. Furthermore, recent studies suggest that the transcription factor GATA-2 is one component that binds Scl enhancer region. Thus, our aim is to further understand mechanisms controlling Flk-1 and Scl gene expression. Specific aim II. Examine the role of BMP-4 mediated signals in hemangioblast development. Our experiments indicate that BMP-4 is critical for the generation of Flk-1 + cells. What is not clear from our current studies is whether BMP-4 mediated signals are still required after the Flk-I + cell stage in establishing hematopoiesis. Our specific aim is to utilize Flk-l-cre mice and conditional Alk-3 (BMP-4 receptor) knockout mice to determine whether BMP-4 mediated signals are obligatory for the generation of the hemangioblast within the Flk-1 + cells. Specific aim III. Examine the role of BMP-4 mediated signals in hematopoietic and endothelial cell development. Subsequent to hemangioblast development, hematopoietic and endothelial cell lineage commitment commences. Our specific aim is to determine whether BMP-4 mediated signals are required for hematopoietic lineage commitment from the hemangioblast. Scl-cre mice and conditional Alk-3 knockout mice will be utilized.