EphA/ephrin-A interactions during optic nerve regeneration: restoration of topography and regulation of ephrin-A2 expression

Abstract

During visual system development, interactions between Eph tyrosine kinase receptors and their ligands, the ephrins, guide retinal ganglion cell (RGC) axons to their topographic targets in the optic tectum. Here we show that Eph/ephrin interactions are also involved in restoring topography during RGC axon regeneration in goldfish. Following optic nerve crush, EphA/ephrin-A interactions were blocked by intracranial injections of recombinant Eph receptor (EphA3-AP) or phospho-inositol phospholipase-C. Topographic errors with multiple inputs to some tectal loci were detected electrophysiologically and increased projections to caudal tectum demonstrated by RT-97 immunohistochemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the retino-recipient tectal layers were reduced in number compared to controls and their distribution was no longer graded. The findings, supported by in vitro studies, implicate EphA/ephrin-A interactions in restoring precise topography and in regulating ephrin-A2 expression during regeneration.

Introduction

Eph tyrosine kinase receptors and their ligands, the ephrins, are cell-membrane bound proteins with tightly regulated expression that mediate cell–cell interactions both during development and in the adult Drescher, 1997, Mc Laughlin et al., 2003. Eph/ephrins are classified into two families: EphAs bind to glycosylphosphatidylinositol (GPI)-linked ephrin-As and EphBs to transmembrane ephrin-Bs (Flanagan and Vanderhaeghen, 1998). Spatially and chronologically restricted interactions between Eph/ephrin-As and/or Bs control many aspects of development including rhombomere formation and blood vessel patterning Brantley et al., 2002, Conover et al., 2000, Cooke and Moens, 2002, Cooke et al., 2001, Durbin et al., 1998, Wang et al., 1998. In addition, Ephs/ephrins play important roles in the nervous system by controlling development of topographic organisation and aspects of synaptic plasticity throughout life Contractor et al., 2002, Gao et al., 1998, Gerlai, 2002, Marı́n et al., 2001, Rogers et al., 1999, Vanderhaeghen et al., 2000.

In the developing visual system, complementary gradients of Eph/ephrins define the projection of retinal ganglion cell (RGC) axons within the major primary visual centre, the optic tectum (superior colliculus in mammals), fulfilling the predictions of Sperry's chemoaffinity hypothesis Karlstrom et al., 1996, Mc Laughlin et al., 2003, Sperry, 1963, Trowe et al., 1996. Eph/ephrins guide RGC axons and control branch formation Connor et al., 1998, Yates et al., 2001 by interactions that are primarily repulsive (EphA/ephrin-As) or attractive (EphB/ephrin-Bs; Holmberg and Frisén, 2002). Gradients and counter-gradients of EphAs and ephrin-As are expressed along the naso-temporal retinal and rostro-caudal tectal axes Brennan et al., 1997, Cheng et al., 1995, Hornberger et al., 1999; gradients of EphBs and ephrin-Bs define the orthogonal dorso-ventral retinal to medio-lateral tectal axes Hindges et al., 2002, Mann et al., 2002. In addition, Eph/ephrins guide outgrowth and fasciculation of RGC axons within the retina, optic nerve and tract Braisted et al., 1997, Caras, 1997, Marcus et al., 1996, Nakagawa et al., 2000, Sefton et al., 1997.

A key role of Eph/ephrin interactions in the development of topography has been revealed using mutant mice lacking or overexpressing one or more of the Eph/ephrin proteins Brown et al., 2000, Feldheim et al., 2000, Hornberger et al., 1999, Park et al., 1997. RGC axon guidance is also abnormal in wild-type animals following in vivo viral mis-expression of ephrin-A2 in the retina or tectum Hornberger et al., 1999, Nakamoto et al., 1996, or when Eph/ephrin interactions are prevented in vivo (Mann et al., 2002) or in vitro Ciossek et al., 1998, Hornberger et al., 1999, Nakamoto et al., 1996, Winslow et al., 1995. Less is known about the role of Eph/ephrin interactions in the restoration of topography following injury in the normal adult. During optic nerve regeneration in goldfish, specific EphAs and ephrin-As are up-regulated coincident with restoration of retino-tectal topography King et al., 2003, Rodger et al., 2000. The result suggests that, as in development, EphA/ephrin-A interactions are required for the restoration of topography.

To test the hypothesis, we prevented EphA/ephrin-A interactions within the goldfish tectum during optic nerve regeneration and examined subsequent topography. We treated the tectum with recombinant EphA3 protein linked to alkaline phosphatase (EphA3-AP) to mask ephrin-As from endogenous EphA receptors. In other animals, we applied phospho-inositol phospholipase-C (PIPLC) to remove all GPI-linked proteins from cell membranes (Low, 1989). The treatments affect all ephrin-As expressed on RGC axons and tectal cells, including the two main contributors to retino-tectal topography, ephrin-A2 and ephrin-A5; the relative contributions of these molecules to optic nerve regeneration remain unknown. We confirmed that both techniques were successful across the entire tectum by detecting either injected (EphA3-AP) or endogenous (PIPLC injected) alkaline phosphatase activity Cheng et al., 1995, Drawbridge and Steinberg, 2000. Endogenous alkaline phosphatase activity can be used to indicate the effectiveness of the PIPLC treatment since AP is a GPI-linked enzyme (Low, 1989). Errors in RGC axon projections were revealed electrophysiologically; neurofilament immunohistochemistry using RT-97, a monoclonal antibody that preferentially binds to regenerating RGC axons (Velasco et al., 2000), verified that RGC axons had regenerated to retino-recipient tectal layers. In vitro explant cultures using recombinant ephrin-A5-AP supported a role for EphA/ephrin-A interactions in guiding regenerating RGC axons.

We also tested the possibility of an additional function of EphA/ephrin-A interactions. Transcription of many receptor–ligand pairs is regulated by a feedback loop signalling via one or both members of the pair. Examples are the growth factor receptor TrkB that, like EphA, is a member of the tyrosine kinase receptor family, and the NMDA glutamate receptor Frank et al., 1996, Goebel and Poosch, 2001. We propose that similarly, EphA/ephrin-A interactions regulate ephrin-A2 expression in the tectum. The hypothesis is supported by the observation that ephrin-A2 up-regulation coincides with the arrival of regenerating RGC axons (Rodger et al., 2000). Using immunohistochemistry and in situ hybridisation, we show that tectal ephrin-A2 expression is abnormal when endogenous EphA/ephrin-A interactions are blocked during optic nerve regeneration; we examined EphA3-AP-injected fish only since PIPLC treatment directly reduces ephrin-A2 protein levels Cheng et al., 1995, Low, 1989. Some of the results have been presented in abstract form (Vitale et al., 2003).