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细胞体外分化模型系统来解决这个问题。我们最近从体外分化的胚胎干细胞中鉴定出了胚集落形成细胞（BL-CFCs），并证明它们代表了造血细胞和内皮细胞（血管母细胞）长期追求的共同祖细胞。因此，它们是研究调节造血开始的因素和信号的理想选择。清楚地了解导致BL-CFC发育的分子机制将揭示胚胎发育中造血规范的途径。我们提出以下具体目标：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，这是造血细胞和内皮细胞的共同祖细胞。我们的数据表明Flk-1+细胞是异质的，Flk-1+SCL+双阳性细胞代表成血管细胞。通过对胚状体（EB）细胞或EB切片的免疫组化，我们将研究Flk-1+细胞是否真的是异质的。我们将进一步确定在血管母细胞上独特表达的标记。这些知识将帮助我们了解成血管细胞的发育是如何被调节的。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）在成血管细胞的发育中起着关键作用。首先，bFGF促进BL-CFCs和Flk-1+细胞的生成。其次，fgfr-1，一种bFGF受体，基因靶向的胚胎干细胞缺乏成血管和造血分化，在体外分化的fgfr-1-/-胚胎干细胞中flk-1和scl的表达都大大降低。因此，对bfgf介导的BL-CFC生成的研究应该能让我们了解成血管细胞的发育是如何被调节的。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的表达对于成血管细胞的发育是否充分和必要。我们还将验证成血管细胞发育也需要bFGF中胚层诱导信号的激活这一假设。这一信息将阐明bfgf介导的BL-CFC发展的分子机制。特异性目的3：我们将研究bFGF、激活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，bFGF和激活A单独或共同积极调节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bFGF、激活素A和骨形态发生蛋白（BMPs）的相互作用影响。我们还将描述携带激活素A或BMP介导信号相关基因靶向突变的胚胎干细胞。这一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.