Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance

https://doi.org/10.1016/j.mcn.2005.05.005Get rights and content

Abstract

Growth cones use cues in their environment in order to grow in a directed fashion to their targets. In Xenopus laevis, fibroblast growth factors (FGFs) participate in retinal ganglion cell (RGC) axon guidance in vivo and in vitro. The main intracellular signaling cascades known to act downstream of the FGF receptor include the mitogen-activated protein kinase (MAPK), phospholipase Cγ (PLCγ) and phosphotidylinositol 3-kinase (PI3K) pathways. We used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF-2-stimulated RGC axon extension and chemorepulsion. The MAPK, PI3K and PLCγ pathways were blocked by U0126, LY249002 and U73122, respectively. D609 was used to test a role for the phosphotidylcholine–PLC (PC–PLC) pathway. We determined that the MAPK and two PLC pathways are required for FGF-2 to stimulate RGC neurite extension in vitro, but the response of axons to FGF-2 applied asymmetrically to the growth cone depended only on the PLC pathways.

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

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