Identification and characterization of FGF target genes

虽然我们可以很容易地识别FGF靶基因，但为了解决我们对基因对FGF信号作出反应的原因的知识不足，我们已经启动了一个项目，从分子上定义导致FGF靶基因对FGF信号作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，这两个已被充分表征的FGF靶基因编码调节FGF信号通路的蛋白质。在我们的初步研究中，我们已经确定了源自Sprouty2的序列，该序列驱动胚胎FGF信号传导关键区域的表达。利用ZIA BC 010338项目（Fgf信号在脊椎动物发育中的作用）中描述的突变体，我们确定了在发育中的小鼠胚胎中，由这些序列驱动的报告基因表达对Fgf信号有反应。转染实验表明，这些序列在培养细胞中也对FGF有反应。现在的目标是a)确定最小的FGF b)确定结合这些序列的反式激活因子，使用基于dna的技术。我们还将使用这些元素作为阳性对照，定义一个细胞培养系统来测定FGF的反应性。这个系统将在我们项目的第二部分中用于筛选新的文库，以确定小鼠基因组中FGF反应性的区域。我们将始终通过体内实验验证任何体外见解，从而提供一种强有力的方法来保证我们数据的重要性。

While we can readily identify FGF targets genes, to address the deficiency in our knowledge of what why genes respond to FGF signaling, 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. 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 FGF signals, in the developing mouse embryo. Transfection experiments reveal that these sequences are also FGF responsive in culture cells. Now the goals are to a) determine the minimal FGF 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.