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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.

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Spatial and temporal d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos. (A & B) Lateral views of d2EGFP mRNA expression at Bud stage and 24 hpf. (C-P) d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos, stages are indicated in each panel. At bud stage (C & D), d2EGFP is detected in the hindbrain (r3/r4, yellow arrowhead) and within the caudal region in the DFCs. (E, G & K) From 8- to 14-somite stages, lateral views show expression of d2EGFP in cells lining Kupffer's vesicle, within r4 (r4, yellow arrowhead) and the mid-hindbrain boundary (mhb, red arrowhead). (F & H) Dorsal views show high d2EGFP expression within the MHB, r4 and the anterior lateral plate mesoderm (alpm, red brackets). (H) At 10-somite stage initial d2EGFP expression is detected within the trigeminal ganglia (tg, blue arrow). (I & J) 24 hpf embryo showing d2EGFP expression in the MHB, trigeminal ganglia, dorsal retina (rt, white arrow) and pharyngeal endoderm (pe, yellow bracket). (K & L) 14 and 20-somite stage embryo highlighting the expression of d2EGFP in Kupffer's vesicle. Higher magnifications are show in (K' & L'). (M) Trunk region shows d2EGFP expression within the dorsal spinal cord neurons (spn, white arrow) at 24 hpf. (N) At 50 hpf expression is noted in the MHB, trigeminal ganglia, pharyngeal endoderm and otic vesicle (ot, blue bracket). (O) Ventral view of 50 hpf, showing d2EGFP expression in the jaw (white bracket). (P) At 56 hpf, strong expression in noted in the trigeminal ganglia, the jaw and also in neurons within the dorsal diencephalon.
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Figure 2: Spatial and temporal d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos. (A & B) Lateral views of d2EGFP mRNA expression at Bud stage and 24 hpf. (C-P) d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos, stages are indicated in each panel. At bud stage (C & D), d2EGFP is detected in the hindbrain (r3/r4, yellow arrowhead) and within the caudal region in the DFCs. (E, G & K) From 8- to 14-somite stages, lateral views show expression of d2EGFP in cells lining Kupffer's vesicle, within r4 (r4, yellow arrowhead) and the mid-hindbrain boundary (mhb, red arrowhead). (F & H) Dorsal views show high d2EGFP expression within the MHB, r4 and the anterior lateral plate mesoderm (alpm, red brackets). (H) At 10-somite stage initial d2EGFP expression is detected within the trigeminal ganglia (tg, blue arrow). (I & J) 24 hpf embryo showing d2EGFP expression in the MHB, trigeminal ganglia, dorsal retina (rt, white arrow) and pharyngeal endoderm (pe, yellow bracket). (K & L) 14 and 20-somite stage embryo highlighting the expression of d2EGFP in Kupffer's vesicle. Higher magnifications are show in (K' & L'). (M) Trunk region shows d2EGFP expression within the dorsal spinal cord neurons (spn, white arrow) at 24 hpf. (N) At 50 hpf expression is noted in the MHB, trigeminal ganglia, pharyngeal endoderm and otic vesicle (ot, blue bracket). (O) Ventral view of 50 hpf, showing d2EGFP expression in the jaw (white bracket). (P) At 56 hpf, strong expression in noted in the trigeminal ganglia, the jaw and also in neurons within the dorsal diencephalon.

Mentions: Reporter gene expression was also analysed at later stages by in situ hybridisation performed to detect the presence of d2EGFP mRNA. At bud stage (10 hpf), d2EGFP transcripts can be detected within the posterior ventral domain and within the presumptive hindbrain, where fgf8 expression has been noted (Figure 2A) [14,30,31]. By 24 hpf, d2EGFP mRNA is detected within the MHB, pharyngeal arches, otic vesicle, retina, optic stalk and dorsal diencephalon (Figure 2B). The ligands fgf8, fgf17 and fgf3, and FGFRs are known to be expressed within these same domains, suggesting that mRNA expression of this reporter gene is under the control of FGF signalling [14,23,25,26,30,31]. In comparison to the fluorescent protein expression, d2EGFP transcripts can be detected in a much wider domain and also more prominent (compare Figure 2A to 2C and Figure 2B to 2I). One reason for the discrepancy can be attributed to the threshold level required for visualisation of d2EGFP protein as compared to the detection of transcripts by in situ hybridisation.


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

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

Spatial and temporal d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos. (A & B) Lateral views of d2EGFP mRNA expression at Bud stage and 24 hpf. (C-P) d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos, stages are indicated in each panel. At bud stage (C & D), d2EGFP is detected in the hindbrain (r3/r4, yellow arrowhead) and within the caudal region in the DFCs. (E, G & K) From 8- to 14-somite stages, lateral views show expression of d2EGFP in cells lining Kupffer's vesicle, within r4 (r4, yellow arrowhead) and the mid-hindbrain boundary (mhb, red arrowhead). (F & H) Dorsal views show high d2EGFP expression within the MHB, r4 and the anterior lateral plate mesoderm (alpm, red brackets). (H) At 10-somite stage initial d2EGFP expression is detected within the trigeminal ganglia (tg, blue arrow). (I & J) 24 hpf embryo showing d2EGFP expression in the MHB, trigeminal ganglia, dorsal retina (rt, white arrow) and pharyngeal endoderm (pe, yellow bracket). (K & L) 14 and 20-somite stage embryo highlighting the expression of d2EGFP in Kupffer's vesicle. Higher magnifications are show in (K' & L'). (M) Trunk region shows d2EGFP expression within the dorsal spinal cord neurons (spn, white arrow) at 24 hpf. (N) At 50 hpf expression is noted in the MHB, trigeminal ganglia, pharyngeal endoderm and otic vesicle (ot, blue bracket). (O) Ventral view of 50 hpf, showing d2EGFP expression in the jaw (white bracket). (P) At 56 hpf, strong expression in noted in the trigeminal ganglia, the jaw and also in neurons within the dorsal diencephalon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Spatial and temporal d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos. (A & B) Lateral views of d2EGFP mRNA expression at Bud stage and 24 hpf. (C-P) d2EGFP expression in Tg(Dusp6:d2EGFP)pt6 embryos, stages are indicated in each panel. At bud stage (C & D), d2EGFP is detected in the hindbrain (r3/r4, yellow arrowhead) and within the caudal region in the DFCs. (E, G & K) From 8- to 14-somite stages, lateral views show expression of d2EGFP in cells lining Kupffer's vesicle, within r4 (r4, yellow arrowhead) and the mid-hindbrain boundary (mhb, red arrowhead). (F & H) Dorsal views show high d2EGFP expression within the MHB, r4 and the anterior lateral plate mesoderm (alpm, red brackets). (H) At 10-somite stage initial d2EGFP expression is detected within the trigeminal ganglia (tg, blue arrow). (I & J) 24 hpf embryo showing d2EGFP expression in the MHB, trigeminal ganglia, dorsal retina (rt, white arrow) and pharyngeal endoderm (pe, yellow bracket). (K & L) 14 and 20-somite stage embryo highlighting the expression of d2EGFP in Kupffer's vesicle. Higher magnifications are show in (K' & L'). (M) Trunk region shows d2EGFP expression within the dorsal spinal cord neurons (spn, white arrow) at 24 hpf. (N) At 50 hpf expression is noted in the MHB, trigeminal ganglia, pharyngeal endoderm and otic vesicle (ot, blue bracket). (O) Ventral view of 50 hpf, showing d2EGFP expression in the jaw (white bracket). (P) At 56 hpf, strong expression in noted in the trigeminal ganglia, the jaw and also in neurons within the dorsal diencephalon.
Mentions: Reporter gene expression was also analysed at later stages by in situ hybridisation performed to detect the presence of d2EGFP mRNA. At bud stage (10 hpf), d2EGFP transcripts can be detected within the posterior ventral domain and within the presumptive hindbrain, where fgf8 expression has been noted (Figure 2A) [14,30,31]. By 24 hpf, d2EGFP mRNA is detected within the MHB, pharyngeal arches, otic vesicle, retina, optic stalk and dorsal diencephalon (Figure 2B). The ligands fgf8, fgf17 and fgf3, and FGFRs are known to be expressed within these same domains, suggesting that mRNA expression of this reporter gene is under the control of FGF signalling [14,23,25,26,30,31]. In comparison to the fluorescent protein expression, d2EGFP transcripts can be detected in a much wider domain and also more prominent (compare Figure 2A to 2C and Figure 2B to 2I). One reason for the discrepancy can be attributed to the threshold level required for visualisation of d2EGFP protein as compared to the detection of transcripts by in situ hybridisation.

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