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BACKGROUND INFORMATION: The fibroblast growth factor (FGF) signalling system of vertebrates is complex. In common with other vertebrates, secreted FGF ligands of the amphibian Xenopus signal through a family of four FGF receptor tyrosine kinases (fgfr1, 2, 3 and 4). A wealth of previous studies has demonstrated important roles for FGF signalling in regulating gene expression during cell lineage specification in amphibian development. In particular, FGFs have well-established roles in regulating mesoderm formation, neural induction and patterning of the anteroposterior axis. However, relatively little is known regarding the role of individual FGFRs in regulating FGF-dependent processes in amphibian development. In this study we make use of synthetic drug inducible versions of Xenopus Fgfr1, 2 and 4 (iFgfr1, 2 and 4) to undertake a comparative analysis of their activities in the tissues of the developing embryo.
RESULTS: We find that Xenopus Fgfr1 and 2 have very similar activities. Both Fgfr1 and Fgfr2 are potent activators of MAP kinase ERK signalling, and when activated in the embryo during gastrula stages regulate similar cohorts of transcriptional targets. In contrast, Fgfr4 signalling in naïve ectoderm and neuralised ectoderm activates ERK signalling only weakly compared to Fgfr1/2. Furthermore, our analyses indicate that in Xenopus neural tissue the Fgfr4 regulated transcriptome is very different from that of Fgfr1.
CONCLUSION AND SIGNIFICANCE: We conclude that signalling downstream of Fgfr1 and 2 regulates similar processes in amphibian development. Interestingly, many of the previously identified canonical transcriptional targets of FGF regulation associated with germ layer specification and patterning are regulated by Fgfr1/Fgfr2 signalling. In contrast, the downstream consequences of Fgfr4 signalling are different, although roles for Fgfr4 signalling in lineage specification and anteroposterior patterning are also indicated.
Figure 1. Schematic diagram of iFgfrs compared to endogenous FGFRs
Panel (A) is a schematic diagram of a wild type FGFR. FGF ligands bind to extracellular Igâlike domains. Receptor monomers are drawn into close proximity, and transphosphorylation of the kinase domains (red) activate downstream FGF signalling pathways. Panel (B) shows an iFgfr tethered to the cell membrane by a myristoylation domain. The 1 µM AP20187 added to culture medium binds to the FKBPv domain, bringing the kinase domains as in (A). The HA tag tethered to the FKBPv domain enables immunodetection of the construct.
Figure 2. Effects of iFgfr activation on MAP kinase signaling
(A) Western blot showing levels of diphosphoâ€ERK (dpERK) and total ERK in animal cap explants from embryos injected with 20 pg iFgfr1, iFgfr2 or iFgfr4 mRNA. Explants were removed at blastula stage 8 and treated with AP20187 for 2 h. (B) Whole mount immunohistochemical detection (vegetal view) of dpERK in the marginal zone of a gastrula stage 10 control embryo. (C) Whole mount immunohistochemical detection (animal view) of dpERK in animal hemisphere cells of gastrula stage 10 control embryos and embryos injected with 20pg iFgfr1, iFgfr2 or iFgfr4 mRNA (plus or minus 1 µM AP20187 treatment from blastula stage 8). Percentages of explants from a representative experiment exhibiting the presented phenotype are indicated.
Figure 3. Effects of iFgfr activation on tissue morphogenesis
Panel (A) compares the appearance of animal caps from embryos injected with 20pg ifgfr1, ifgfr2 or ifgfr4 mRNA ± 1 µM AP20187. AP20187 treatments occurred from blastula stage 8 until late neurula stage 18. Percentages of explants from a representative experiment exhibiting the presented phenotype are indicated, ifgfr1, n = 9, ifgfr2 n = 12, ifgfr4, n = 11. Panel (B) compares the appearance at the tailbud stage of embryos injected with 20pg ifgfr1, ifgfr2 or ifgfr4 mRNA (±1 µM AP20187). AP20187 treatment was continuous from gastrula stage 10.5. Percentages of embryos from a representative experiment exhibiting the presented phenotype are indicated, iFgfr1, n = 20, iFgfr2 n = 15, iFgfr4, n = 20.
Figure 4. Effects of iFgfr1 and iFgfr2 activation on the transcriptome of gastrula stage embryos
Scatterplots of log2 gene expression levels in late gastrula stage 13 embryos were generated using the Affymetrix microarray platform. Data were filtered with a cutâ€off to exclude array features with expression levels <50. Data points in the white zone proximal to the black line (y = x) have less than twofold change in expression. Data points in the green zone have more than twofold increase in expression relative to control (xâ€axis). Data points in the red zone indicate more than twofold decrease in expression relative to control. Panel (A) compares gene expression in control uninjected embryos and uninjected embryos treated with 1 µM AP20187 from early gastrula stage 10.5. Panel (B) compares gene expression in embryos injected with 20pg ifgfr1 or ifgfr2 mRNA in the absence of AP20187 treatment (uninduced). Panels (C) and (D) compare gene expression in embryos injected with either 20pg iFgfr1 (C) or iFgfr2 mRNA (D) cultured in the absence (uninduced) or presence of 1 µM AP20187 from stage 10.5 (induced). Panel (E) is a Venn diagram showing the overlap of the gene sets up†and downregulated by more than or equal to twofold following activation of iFgfr1 and iFgfr2 signalling during gastrula stages.
Figure 5. Effects of iFgfr1 and iFgfr4 activation in neural tissue
Panel (A) compares the appearance of control uninjected tailbud stage embryos and embryos injected into the two dorsoâ€animal blastomeres at the eightâ€cell stage with 20 pg ifgfr1 or ifgfr4 mRNA cultured in the absence (uninduced) or presence of 1 µM AP20187 from stage 10.5 (induced). Higher magnification images of disrupted eye development are shown inset. Black arrow indicates missing lens. White arrow indicates disrupted pigmentation in the retina. (B) Western blot showing levels of phosphoâ€Smad1, 5, 8 (pSmad1/5/8), diphosphoâ€ERK (dpERK) and total ERK animal cap explants at early neurula stage 15. Explants were taken from control uninjected embryos and embryos injected with 20 pg ifgfr1 mRNA or 50 pg noggin mRNA or coinjected with 20 pg ifgfr1 mRNA and 50 pg noggin mRNA. Explants from embryos injected with ifgfr1 were cultured in the absence (uninduced) or presence of 1 µM AP20187 from stage 10.5 (induced). (C) Western blot analysing levels of diphosphoâ€ERK (dpERK) and total ERK levels animal cap explants from embryos injected with 20 pg ifgfr1 mRNA or ifgfr4 mRNA. Explants were cultured in the absence (uninduced) or presence of 1 µM AP20187 from stage 10.5 (induced) for 3 h.
Figure 6. Effects of iFgfr1 and iFgfr4 activation on the transcriptome of neuralised tissue explants
Scatterplots of log2 expression gene expression levels in neuralised animal cap explants generated from RNAâ€seq analysis data. Data were filtered with a cutâ€off to exclude identified gene models with expression levels <30 (FKPM). Data points in the white zone, proximal to the black line (y = x) have less than twofold change in expression. Data points in the green zone have a more than twofold increase in expression relative to control (xâ€axis). Data points in the red zone indicate more than twofold decrease in expression relative to control. Panel (A) compares gene expression in explants from embryos injected with 20 pg ifgfr1 or ifgfr4 mRNA in the absence of AP20187 treatment (uninduced). Panels (B) and (C) compare gene expression in explants from embryos injected with 20 pg ifgfr1 (B) or ifgfr4 mRNA (C) cultured in the absence (uninduced) or presence of 1 µM AP20187 from stage 10.5 for 3 h (induced). Panel (D) is a Venn diagram showing the overlap of gene sets up and down regulated by more than or equal to twofold following activation of iFgfr1 and iFgfr4 signalling in neuralised animal caps explants.
