Abstract
BackgroundFibroblast 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.ResultsExpression 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.ConclusionThe 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.
Highlights
Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes
These FGF reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the receptor tyrosine kinases (RTKs) family
Since FGF signalling during embryogenesis is dynamic, we reasoned that a destabilized form of EGFP would likely recapitulate the endogenous expression of dusp6 mRNA. 1-cell stage zebrafish embryos were co-injected with the transgenic DNA construct and I-Sce 1 meganuclease as described by Thermes et al to generate transgenic lines [37]
Summary
Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes. Understanding the temporal and spatial activity of signalling peptides is key to determining the role for these factors in controlling cellular fates. Fibroblast Growth Factors (FGFs), a family of secreted glycoproteins, perform crucial functions that include the establishment of embryo polarity, the formation of organizing centres, and the induction of limb outgrowth [1,2,3]. These ligands are expressed in discrete domains during development and their actions are restricted to cells that express integral membrane proteins that can bind FGFs [2,4]. How FGFs control gene expression and the nature of the genes that they regulate during development is still not completely established
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