Limits...
Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.

Molina GA, Watkins SC, Tsang M - BMC Dev. Biol. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA. gam20@pitt.edu <gam20@pitt.edu>

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH

Related in: MedlinePlus

Expression of fli1 in intersegmental vessels in indolinone treated embryos. (A-H) Lateral trunk views of fli mRNA expression within the ISV. (A) DMSO control shows vessel sprouts at 28 hpf. (B-G) Embryos treated with the compounds indicated show loss of ISV sprouts, while in (H) SU6656 did not affect these vessels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1904198&req=5

Figure 8: Expression of fli1 in intersegmental vessels in indolinone treated embryos. (A-H) Lateral trunk views of fli mRNA expression within the ISV. (A) DMSO control shows vessel sprouts at 28 hpf. (B-G) Embryos treated with the compounds indicated show loss of ISV sprouts, while in (H) SU6656 did not affect these vessels.

Mentions: To confirm our findings that VEGFR inhibitors did indeed block intersegmental vessel outgrowth, we performed in situ hybridisation studies to detect the presence of these vessels through fli1 expression. Treatment of embryos with 1 μM Oxindole 1, 10 μM SU5416, 20 μM SU4312, 1 μM SU5402, or with 20 μM SU11652 prevented ISV formation as determined by loss of fli1 expression in the intersegmental sprouts (Figure 8B–G). Again these results confirm that SU5402 can block VEGFR signalling in the zebrafish and is not a specific inhibitor of FGFRs. In contrast, expression of fli1 within the ISV was unaffected in embryos treated with 10 μM SU6656, confirming the notion that Src kinases are not required in ISV outgrowth (Figure 8H).


Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.

Molina GA, Watkins SC, Tsang M - BMC Dev. Biol. (2007)

Expression of fli1 in intersegmental vessels in indolinone treated embryos. (A-H) Lateral trunk views of fli mRNA expression within the ISV. (A) DMSO control shows vessel sprouts at 28 hpf. (B-G) Embryos treated with the compounds indicated show loss of ISV sprouts, while in (H) SU6656 did not affect these vessels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904198&req=5

Figure 8: Expression of fli1 in intersegmental vessels in indolinone treated embryos. (A-H) Lateral trunk views of fli mRNA expression within the ISV. (A) DMSO control shows vessel sprouts at 28 hpf. (B-G) Embryos treated with the compounds indicated show loss of ISV sprouts, while in (H) SU6656 did not affect these vessels.
Mentions: To confirm our findings that VEGFR inhibitors did indeed block intersegmental vessel outgrowth, we performed in situ hybridisation studies to detect the presence of these vessels through fli1 expression. Treatment of embryos with 1 μM Oxindole 1, 10 μM SU5416, 20 μM SU4312, 1 μM SU5402, or with 20 μM SU11652 prevented ISV formation as determined by loss of fli1 expression in the intersegmental sprouts (Figure 8B–G). Again these results confirm that SU5402 can block VEGFR signalling in the zebrafish and is not a specific inhibitor of FGFRs. In contrast, expression of fli1 within the ISV was unaffected in embryos treated with 10 μM SU6656, confirming the notion that Src kinases are not required in ISV outgrowth (Figure 8H).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA. gam20@pitt.edu <gam20@pitt.edu>

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus