Identification and characterization of FGF target genes

为了解决我们对FGF靶基因的定义知识的不足，我们启动了一个项目，从分子上定义导致FGF靶基因对FGF信号做出反应的顺式作用元件。该项目的一个分支涉及表征调节Sprouty2和Sprouty4表达的元件，这两个充分表征的FGF靶基因编码调节FGF信号通路的蛋白质。我们已经确定，这些Sprouty基因在我们的Bmpr突变体中也响应BMP信号传导，该突变体在项目ZIA BC 010518（肢体发育期间BMP和FGF信号传导的作用）中描述。因此，我们也在探索Sprouty基因中的关键控制元件可能受BMP和FGF信号之间的相互作用调节的想法。在我们的初步研究中，我们已经定义了来自Sprouty2的序列，这些序列驱动胚胎FGF信号传导关键区域的表达。使用项目ZIA BC 010338（脊椎动物发育中FGF信号传导的作用）中描述的突变体，我们已经确定由这些序列驱动的报告基因表达对体内FGF信号有响应。转染实验表明，这些序列在培养细胞中也是FGF响应性的。现在的目标是a）确定最小的FGF和BMP反应元件B）使用基于DNA的技术确定与这些序列结合的反式激活因子。我们还将使用这些元素作为阳性对照，定义细胞培养系统，在其中测定FGF反应性。这样的系统将被用来，在我们的第二个手臂的这个项目，筛选新的库，以确定区域的FGF反应在小鼠基因组。我们将始终通过体内实验来验证任何体外见解，从而提供一种强大的方法来保证我们数据的重要性。

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.