Hematopoietic Commitment: Molecular Mechanisms

描述(由申请者提供)：我们的长期目标是了解在发育中的胚胎中造血系统是如何建立的。我们对体外分化的胚胎干细胞的研究表明，造血系统是通过不同的顺序产生的表达Flk-1和SCL的细胞来建立的。Flk-I+SCL-细胞最早出现在发育中的拟胚体(EBS，ES细胞的体外分化后代)。在Flk-1+SCL-细胞中，scl基因被激活，从而产生Flk-1+SCL+细胞。血管母细胞群是造血细胞和内皮细胞的共同前体，在这些细胞中得到了丰富。在Flk-I+SCL+细胞中，Flk-1表达下调，最终产生Flk-I-SCL+造血祖细胞。无血清ES细胞体外分化模型证实骨形态发生蛋白(BMP)-4在Flk-1+和SCL+细胞的产生中起关键作用。许多研究表明BMP-4在造血发育中的重要性。然而，没有一项研究考察BMP-4发挥作用的确切发育阶段。我们的建议是进一步确定控制Flk-1和scl表达的分子机制，并确定BMP-4在哪个确切的发育阶段发挥作用，以建立造血系统。 具体目的I：进一步表征与Flk-1+和SCL+细胞生成有关的分子事件。我们的研究表明，BMP-4激活MAP激酶和Smadl通路对Flk-1+细胞的产生至关重要。此外，最近的研究表明，转录因子GATA-2是结合scl增强子区的一个成分。因此，我们的目的是进一步了解Flk-1和scl基因表达的调控机制。 特定目的II.研究BMP-4介导的信号在血管母细胞发育中的作用。我们的实验表明，BMP-4对Flk-1+细胞的产生至关重要。我们目前的研究尚不清楚的是，在Flk-I+细胞阶段之后，是否仍然需要BMP-4介导的信号来建立造血。我们的具体目的是利用Flk-L-cre小鼠和条件性ALK-3(BMP-4受体)基因敲除小鼠来确定BMP-4介导的信号是否对Flk-1+细胞内血管母细胞的生成是必需的。 特定目的III.研究BMP-4介导的信号在造血和内皮细胞发育中的作用。在血管母细胞发育之后，造血细胞和内皮细胞的谱系承诺开始。我们的具体目的是确定BMP-4介导的信号是否需要来自血管母细胞的造血系承诺。将利用SCL-cre小鼠和条件性ALK-3基因敲除小鼠。

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.