Identification and characterization of FGF target genes

为了解决我们对什么定义成纤维细胞生长因子靶基因的认识不足，我们启动了一个项目，从分子上定义导致成纤维细胞生长因子靶基因对成纤维细胞生长因子信号做出反应的顺式作用元件。该项目的一部分涉及表征调节Sprouty2和Sprouty4表达的元件，这是两个特征良好的成纤维细胞生长因子靶基因，它们编码调节成纤维细胞生长因子信号通路的蛋白质。我们已经确定，在Zia BC 010518项目(骨形态发生蛋白和成纤维细胞生长因子信号在肢体发育中的作用)中描述的我们的Bmpr突变体中，这些芽胞基因也对骨形态发生蛋白信号做出反应。因此，我们也在探索Sprouty基因中的关键控制元件，这些元件可能受BMP和成纤维细胞生长因子信号之间的相互作用调节。在我们的初步研究中，我们定义了来源于Sprouty2的序列，这些序列驱动着胚胎成纤维细胞生长因子信号的关键区域的表达。利用Zia BC 010338项目中描述的突变体(成纤维细胞生长因子信号在脊椎动物发育中的作用)，我们已经确定了由这些序列驱动的报告基因表达对体内成纤维细胞生长因子信号有反应。转染实验表明，这些序列在培养细胞中也对成纤维细胞生长因子有反应。现在的目标是a)确定最小的成纤维细胞生长因子和骨形态发生蛋白反应元件b)确定与这些序列结合的反式活性因子，使用基于DNA的技术。我们还将使用这些元素作为阳性对照，定义一种细胞培养系统，在该系统中检测成纤维细胞生长因子的反应性。这样的系统将在我们这个项目的第二个手臂中用于筛选新的文库，以确定小鼠基因组中成纤维细胞生长因子的反应性区域。我们将始终通过活体实验验证任何体外洞察，从而提供一种强有力的方法，确保我们数据的重要性。

To address the deficiency in our knowledge of what defines an FGF target gene, we have initiated a project to molecularly define the cis-acting elements that cause FGF targets genes to respond to FGF signals. One arm of this project involves characterizing the elements that regulate the expression of Sprouty2 and Sprouty4, two well-characterized FGF target genes that encode proteins that regulate the FGF signaling pathway. We have determined that these Sprouty genes also respond to BMP signaling in our Bmpr mutants that are described in project ZIA BC 010518 (Role of BMP and FGF signaling during limb development). Therefore we are also exploring the idea that the key control elements in Sprouty genes that may be regulated by an interaction between BMP and FGF signaling. In our preliminary studies we have defined sequences derived from Sprouty2 that drive expression in key regions of embryonic FGF signaling. Using mutants described in project ZIA BC 010338 (The Role of Fgf Signaling in Vertebrate Development ) we have determined that reporter gene expression, driven by these sequences, is responsive to in vivo FGF signals. Transfection experiments reveal that these sequences are also FGF responsive in culture cells. Now the goals are to a) determine the minimal FGF and BMP responsive elements b) determine the trans-activing factors that bind to these sequences, using DNA-based techniques. We will also use these elements, as positive controls, define a cell culture system in which to assay FGF responsiveness. Such a system will be used, in our second arm of this project, to screen novel libraries to define regions of FGF responsiveness in the mouse genome. We will always validate any in vitro insights with in vivo experiments, thus providing a powerful approach that should guarantee the significance of our data.