HEMATOPOIETIC COMMITMENT--MOLECULAR MECHANISMS

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。 我们利用小鼠胚胎干（ES）细胞的体外分化模型系统来解决这个问题。 我们最近已经确定了胚集落形成细胞（BL-CFCs）在体外分化的ES细胞，并证明他们代表了长期追求的造血和内皮细胞，血管母细胞的共同祖细胞。 因此，它们是研究造血发生的调节因子和信号的理想选择。 对导致BL-CFC发育的分子机制的清楚理解应该揭示发育胚胎中造血特化的途径。 我们提出了以下具体目标：具体目标1：我们将进一步表征Flk- 1+细胞以定义成血管细胞。 我们已经证明Flk-1+细胞含有成血管细胞，一种造血和内皮细胞的共同祖细胞。 我们的数据表明，Flk- 1+细胞是异质性的，Flk-1+SCL+双阳性细胞代表成血管细胞。 通过利用免疫组织化学对总胚状体（EB）细胞或EB切片，我们将调查是否Flk-1+细胞是真正的异质性。 我们将进一步鉴定成血管细胞上独特表达的标记物。 这些知识将帮助我们了解成血管细胞的发育是如何调节的。 具体目标2：我们将确定bFGF介导的BL-CFC生成的分子机制。 我们的数据表明碱性成纤维细胞生长因子（bFGF）在成血管细胞发育中发挥着关键作用。 首先，bFGF增强BL-CFC和Flk-1+细胞的生成。 第二，fgfr-1（一种bFGF受体）基因靶向的ES细胞在成血管细胞和造血分化中是缺陷的，并且在体外分化的fgfr-1-/- ES细胞中flk-1和scl的表达都大大降低。 因此，bFGF介导的BL-CFC生成的研究应该给我们一个线索，如何成血管细胞的发展受到调节。为了确定bFGF介导的成血管细胞生成的分子基础，我们将首先确定Flk-1和/或SCL的表达是否是成血管细胞发育所必需的。 我们还将测试的假设，激活bFGF中胚层诱导信号也需要成血管细胞的发展。 这些信息将阐明bFGF介导的BL-CFC发展的分子机制。 具体目标3：我们将研究bFGF、激活素A、BMP-2或BMP-4是否可以共同调节成血管细胞的发育。 我们的数据表明，碱性成纤维细胞生长因子和激活素A，单独或一致，积极调节BL-CFC的产生。为了进一步确定导致成血管细胞发育的分子机制，我们将确定BL-CFC的发育是否受到bFGF、激活素A和骨形态发生蛋白（BMP）的相互作用。 我们还将表征在激活素A或BMP介导的信号中涉及的基因中携带靶向突变的ES细胞。这些信息将进一步增强我们对胚胎内造血发育的理解。

Our long-term goal is to understand how the hematopoietic system is established in the developing embryo. We utilize an in vitro differentiation model system of mouse embryonic stem (ES) cells to address this issue. We have recently identified blast colony forming cells (BL-CFCs) from in vitro differentiated ES cells and demonstrated that they represent the long pursued common progenitor of hematopoietic and endothelial cells, the hemangioblast. Therefore, they are ideal for studying factors and signals regulating the onset of hematopoiesis. A clear understanding of the molecular mechanisms leading to BL-CFC development should unveil the pathway of hematopoietic specification in the developing embryo. We propose the following specific goals: Specific Aim 1: We will further characterize Flk- 1+ cells to define the hemangioblast. We have shown the Flk-1+ cells contain hemangioblasts, a common progenitor of hematopoietic and endothelial cells. Our data suggest that Flk- 1+ cells are heterogeneous and the Flk-1+SCL+ double positive cells represent hemangioblasts. By utilizing immunohistochemistry on total embryoid body (EB) cells or EB sections, we will investigate if Flk-1+ cells are truly heterogeneous. We will further identify markers uniquely expressed on hemangioblasts. This knowledge will help us understand how hemangioblast development is regulated. Specific Aim 2: We will determine molecular mechanisms involved in bFGF- mediated BL-CFC generation. Our data suggest that basic fibroblast growth factor (bFGF) plays a critical role in hemangioblast development. First, bFGF enhances the generation of BL-CFCs and Flk-1+ cells. Second, fgfr-1, a bFGF receptor, gene targeted ES cells are deficient in hemangioblast and hematopoietic differentiation and the expression of both flk-1 and scl is greatly reduced in in vitro differentiated fgfr-1-/- ES cells. Therefore, studies of bFGF-mediated BL-CFC generation should give us a clue how hemangioblast development is regulated. To define the molecular basis of bFGF-mediated hemangioblast generation, we will first determine if the expression of Flk-1 and/or SCL is sufficient and necessary for hemangioblast development. We will also test the hypothesis that the activation of bFGF mesoderm inductive signals is also required for hemangioblast development. This information will elucidate the molecular mechanisms involved in bFGF-mediated BL-CFC development. Specific Aim 3: We will investigate if bFGF, activin A, BMP-2, or BMP-4 could co-regulate hemangioblast development. Our data indicate that bFGF and activin A, singularly or in concert, positively regulate BL-CFC generation. To further define molecular mechanisms leading to hemangioblast development, we will determine if BL-CFC development is subject to interplay of bFGF, activin A, and bone morphogenetic proteins (BMPs). We will also characterize ES cells carrying targeted mutations in genes involved in activin A or BMP mediated signals. This information will further enhance our understanding of hematopoietic development within the embryo.