Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.
Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.
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DOI：
10.1186/1471-213x-7-62
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发表时间：
2007-06-06
影响因子：
--
通讯作者：
Tsang M
中科院分区：
文献类型：
--
作者：
Molina GA;Watkins SC;Tsang M
成纤维细胞生长因子（FGF）代表了正常发育和生理过程所需的分泌蛋白质的大家族。小鼠和斑马鱼FGF中的突变导致异常胚胎发生和致死。了解这些因子的确切作用的关键是确定它们在胚胎发生过程中的空间和时间活性。双特异性磷酸酶6（dusp 6，也称为Mkp 3）的表达在整个发育过程中由FGF信号传导控制。Dusp 6启动子从斑马鱼中分离，并用于驱动转基因胚胎（Tg（Dusp 6：d2 EGFP））中不稳定的绿色荧光蛋白（d2 EGFP）的表达。d2 EGFP的表达早在受精后4小时（hpf）就在胚胎的未来背侧区域内开始，其中fgf 3和fgf 8最初表达。在后期阶段，在与Fgf配体及其受体的表达相关的结构内检测到d2 EGFP。这包括中脑-后脑边界（MHB）、咽内胚层、耳泡、后脑和库普弗囊泡。d2 EGFP的表达受FGF信号传导的控制，因为用FGF受体（FGFR）抑制剂处理导致d2 EGFP表达的抑制。在市售小分子的中试筛选中，我们评估了转基因株系鉴定吲哚啉酮类内特异性FGF抑制剂的有效性。这些化合物用转基因系Tg（Fli 1：EGFP）y1反筛选，其用作血管内皮生长因子（VEGF）信号传导的间接读出，以确定相关受体酪氨酸激酶（RTK）之间的特异性。从这些测定中，可以确定这些吲哚啉酮对特定RTK信号传导途径的特异性。这使得能够鉴定可以特异性阻断VEGFR或FGFR信号传导途径的化合物。转基因报告斑马鱼系的产生允许直接可视化发育胚胎内的FGF信号传导。这些FGF报告基因转基因系提供了一种筛选特定化合物的工具，该化合物可以区分RTK家族的两个保守成员。
Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis. Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3) is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP) in transgenic embryos (Tg(Dusp6:d2EGFP)). Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf) within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB), pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR) inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFP)y1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF) signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs). From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway. The generation of transgenic reporter zebrafish lines has allowed direct visualization of FGF signalling within the developing embryo. These FGF reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the RTK family.