Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance
Introduction
Growth cones are highly motile sensory structures at the tips of growing axons that guide axons to their targets by responding to external guidance cues in their environment. We have previously shown that in Xenopus laevis, fibroblast growth factors (FGFs) act as guidance cues for the axons of the main output cells of the retina, the retinal ganglion cells (RGCs). RGC growth cones express FGF receptors (FGFRs) and travel through an FGF-2-enriched forebrain en route to their midbrain target, the optic tectum (McFarlane et al., 1995, McFarlane et al., 1996). Moreover, functional FGFRs are required cell autonomously for RGC axons to recognize their target (McFarlane et al., 1996). Finally, FGF-2 can directly repel RGC axons both in vitro and in vivo, which suggests that FGFs chemotropically influence RGC growth cones (Webber et al., 2003). In this paper, we investigated the intracellular signaling pathway(s) downstream of FGF-mediated axon extension and chemorepulsion.
The main intracellular signaling pathways known to act downstream of the receptor tyrosine kinase FGFR include the mitogen-activated protein kinase (MAPK), phospholipase Cγ (PLCγ) and phosphotidylinositol 3-kinase (PI3K) pathways (reviewed by Klint and Claesson-Welsh, 1999). All three of these pathways function in the guidance of Xenopus axons. For instance, Xenopus spinal neuron growth cone turning in response to a netrin-1 gradient is dependent on PLCγ and PI3K co-activation (Ming et al., 1999). Further, the response of RGC growth cones to netrin-1 is regulated by MAPK-dependent changes in protein synthesis at the growth cone (Campbell and Holt, 2003). While the PLCγ pathway is required for FGF-2 to stimulate RGC axon outgrowth both in vitro and in vivo (Lom et al., 1998), we do not know the function of the other pathways in FGF-stimulated axon extension, nor which pathway(s) acts downstream of the chemotropic actions of FGF-2 on RGC growth cones.
To investigate this we used pharmacological inhibitors to block FGFR signaling pathways in in vitro and in vivo preparations designed to examine axon extension and guidance. Specifically, the MAPK, PLCγ and PI3K pathways, as well as a novel pathway downstream of the FGFR, the phosphotidylcholine phospholipase C (PC–PLC) pathway, were targeted. PI3K appeared unnecessary for RGC axon extension, but the other three pathways acted in a convergent fashion to mediate FGF-stimulated axon extension in vitro. In contrast, only the PLC pathways were required for the turning response of growth cones to an FGF-2 gradient.
Section snippets
The MAPK and PC–PLC Pathways are necessary for optic tract extension in vivo
To determine which signaling pathways are involved in optic tract development, pharmacological inhibitors were applied to the developing optic projection using a previously described exposed brain preparation (Chien et al., 1993, Webber et al., 2002). At stage 33/34, the optic tract has crossed the optic chiasm to reach the contralateral forebrain. At this stage, the skin and dura from this side of the brain were removed and the appropriate inhibitors were added to the bathing media. Since the
Discussion
The purpose of this study was to determine which intracellular signaling pathway(s) acts downstream of the FGFR to regulate RGC axon extension and guidance. Our exposed brain experiments, and those of Lom et al. (1998), indicated that RGC axon extension in vivo requires activation of the MAPK, PC–PLC and PLCγ pathways, but possibly not the PI3K pathway. In vitro, our results suggest that the MAPK, PC–PLC and PLCγ pathways converge to mediate an FGF-2-induced increase in RGC neurite extension,
Animals
Eggs obtained from adult female Xenopus laevis injected with human chorionic gonadotrophin (Intervet) were fertilized in vitro to generate embryos. Embryos were kept in 0. 1× Marc's modified ringer's solution (MMR; 0.1 M NaCl, 2 mM KCl, 1 mM MgCl2, 2mM CaCl2, 5 mM HEPES, pH 7.5), with the temperature varying between 14°C and 27°C to control their speed of development. Embryos were staged according to Nieuwkoop and Faber (1994).
Retinal cultures
Eye primordia were dissected from stage 24 Xenopus embryos. For
Acknowledgments
The authors would like to thank J.C. Hocking, K. Atkinson-Leadbeater and Dr. S. Robbins for comments on the manuscript. R. Parker provided excellent technical assistance. The work was funded by an operating grant from the Canadian Institutes of Health Research (CIHR). Infrastructure support was provided by the Canadian Foundation for Innovation, the Neurodegenerative Disease Endowment, the Edwin I. and John Edwin Gustus Endowment and the Arthur Henry and Alice Elizabeth Zoe Fitzgerald
References (66)
- et al.
Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation
Cell Signalling
(2003) - et al.
The TrkB-Shc site signals neuronal survival and local axon growth via MEK and P13-kinase
Neuron
(2000) - et al.
Brain-derived neurotrophic factor increases Ca2+/calmodulin-dependent protein kinase 2 activity in hippocampus
J. Biol. Chem.
(1997) - et al.
Control of cell migration during Caenorhabditis elegans development
Curr. Opin. Cell Biol.
(1999) - et al.
Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones
Neuron
(2003) - et al.
Navigational errors made by growth cones without filopodia in the embryonic Xenopus brain
Neuron
(1993) - et al.
Precocious pathfinding: retinal axons can navigate in an axonless brain
Neuron
(1992) - et al.
Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC
Neuron
(1997) - et al.
Regulation of bidirectional melanosome transport by organelle bound MAP kinase
Curr. Biol.
(2005) - et al.
Cytoskeletal dynamics and transport in growth cone motility and axon guidance
Neuron
(2003)
Rho GTPases in growth cone guidance
Curr. Opin. Neurobiol.
Two cellular inductions involved in photoreceptor determination in the Xenopus retina
Neuron
Guiding neuronal growth cones using Ca2+ signals
Trends Cell Biol.
Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma-dependent pathway
J. Biol. Chem.
Distinct signaling pathways involved in multiple effects of basic fibroblast growth factor on cultured rat hippocampal neurons
Brain Res.
Compound D609 inhibits phorbol ester-stimulated phospholipase D activity and phospholipase C-mediated phosphatidylethanolamine hydrolysis
Biochim. Biophys. Acta
Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?
J. Biol. Chem.
FGF signaling and target recognition in the developing Xenopus visual system
Neuron
Inhibition of FGF receptor activity in retinal ganglion cell axons causes errors in target recognition
Neuron
Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance
Neuron
Phosphatidylcholine-specific phospholipase C regulates thapsigargin-induced calcium influx in human lymphocytes
J. Biol. Chem.
Phosphatidylinositol 3-kinase signaling is involved in neurogenesis during Xenopus embryonic development
J. Biol. Chem.
Involvement of phosphatidylcholine-specific phospholipase C in platelet-derived growth factor-induced activation of the mitogen-activated protein kinase pathway in Rat-1 fibroblasts
J. Biol. Chem.
The mitochondrial-apoptotic pathway is triggered in Xenopus mesoderm cells deprived of PDGF receptor signaling during gastrulation
Dev. Biol.
Role of axons in membrane phospholipid synthesis in rat sympathetic neurons
Neurobiol. Aging
1,10-Phenanthroline phosphorylates (activates) MAP kinase in Xenopus oocytes
Cell Signalling
Fibroblast growth factors redirect retinal axons in vitro and in vivo
Dev. Biol.
NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC
Neuron
Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth
Proc. Natl. Acad. Sci. U. S. A.
Nitric oxide production stimulated by the basic fibroblast growth factor requires the synthesis of ceramide
Ann. N. Y. Acad. Sci.
Hydrolysis of phosphatidylcholine is stimulated by Ras proteins during mitogenic signal transduction
Mol. Cell. Biol.
Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction
Mol. Cell. Biol.
Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction
Development
Cited by (38)
Receptor Tyrosine Kinases and Phosphatases in Neuronal Wiring: Insights From Drosophila
2017, Current Topics in Developmental BiologyCitation Excerpt :The Drosophila FGFs are Branchless (Btl ligand), Thisbe, and Pyramus (the last two are Htl ligands; Huang & Stern, 2005; Kadam, McMahon, Tzou, & Stathopoulos, 2009). In contrast to what has been identified in vertebrate systems where FGFs play crucial roles in the neuronal wiring of diverse systems (Shirasaki, Lewcock, Lettieri, & Pfaff, 2006; Webber, Chen, Hehr, Johnston, & McFarlane, 2005; Webber, Hyakutake, & McFarlane, 2003), only few studies report the function of FGF signaling in Drosophila neuronal wiring. The first observation described a function for the Heartless FGFR in axon guidance.
The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance
2015, Developmental CellCitation Excerpt :As expected, netrin-dependent increases in Trim9+/+ filopodia and excess filopodia in Trim9−/− growth cones were blocked by CytoD (Figure S4B, p < 0.005). To further test the specificity of TRIM9 function in netrin and DCC-dependent filopodia increases, neurons were treated with Fibroblast growth factor 2 (FGF-2), an attractive guidance cue that functions through a distinct signaling pathway (Webber et al., 2005; Zechel et al., 2010). FGF2 increased filopodia density in both Trim9+/+ and Trim9−/− growth cones (p < 0.05, Figures 4A and 4B).
Rho kinase is required to prevent retinal axons from entering the contralateral optic nerve
2015, Molecular and Cellular NeuroscienceCitation Excerpt :At 90–100 μM, the vast majority of embryos (15/17) exhibited the contralateral optic nerve phenotype, as confirmed in transverse vibratome sections (10/10 embryos with > 10 axons in the contralateral optic nerve). Of note, the use of pharmacological inhibitors of other intracellular kinases in the exposed brain preparation does not produce a similar defect (Webber et al., 2005). As a final pharmacological means of manipulating Rock activity to verify the specificity of the effects we observed with the Rock inhibitors, we took advantage of the known negative regulation of Rock by the Protein kinase A (PKA) pathway (Brown et al., 2012; Park et al., 2006; Tkachenko et al., 2011).
Dental pulp stem cells differentiation into retinal ganglion-like cells in a three dimensional network
2015, Biochemical and Biophysical Research CommunicationsCitation Excerpt :FGF2 plays an important role in RGC differentiation, RGC axon growth and axon guidance [27]. It has been shown that FGF2 stimulate neurite extension of RGCs in vitro [28] and is a potent stimulator of axon growth during RGC development, so it is crucial for RGC differentiation [29,30]. FGF2 and Shh activate Pax6, a neural/retinal progenitor marker.
A quantitative approach to the dynamics of neurite sprouting induced by a neurotrophic factor
2009, Journal of Neuroscience MethodsCitation Excerpt :This function is of great relevance in the establishment of the correct connections between the individual components of the nervous system. Basic Fibroblast Growth Factor (bFGF) is one of the most extensively studied neurotrophic factors in this context (Abe and Saito, 2001), and it has been shown to exert different roles according to the specific experimental model: in many sensory and autonomic neurons it acts by promoting neurite growth (Perron and Bixby, 1999; Webber et al., 2005; Zamburlin et al., 2006), while in central neurons it has been shown to inhibit axonal growth (Catapano et al., 2004) or induce growth cone arrest and formation of branches and collaterals (Szebenyi et al., 2001). An equally relevant, although less studied, aspect is the role of the neurotrophic factor in the definition of the neuronal morphology, e.g. of the number of neurites that each neuron produces and subsequently stabilizes.
In vitro differentiation of retinal ganglion-like cells from embryonic stem cell derived neural progenitors
2009, Biochemical and Biophysical Research CommunicationsCitation Excerpt :FGF2 also plays an important role in RGC differentiation, RGC axon growth and axon guidance [16]. It has been shown that FGF2 stimulate neurite extension of RGCs in vitro[17]. There was a 10-fold increase in the retinal ganglion axon growth in the presence of FGF2 suggesting that FGF2 is a potent stimulator of axon growth during RGC development [16].