EphrinA6 on chick retinal axons is a key component for p75NTR-dependent axon repulsion and TrkB-dependent axon branching

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Abstract

A characteristic of the ephrin/Eph family is their capacity for bi-directional signalling. This means that an ephrin, for example, can function either as a ligand for an Eph ‘receptor’, or as a receptor for an Eph ‘ligand’. A system in which this phenomenon is well studied is the retinotectal projection in which the guidance of retinal ganglion cell (RGC) axons to their target area in the tectum is controlled by both Ephs and ephrins expressed in gradients in both the retina and tectum.

Here we have analysed the receptor function of ephrinAs on RGC axons in further detail by focussing on ephrinA6, which is the most strongly expressed ephrinA in the chick retina. EphrinAs are GPI-anchored proteins and therefore require the interaction with transmembrane proteins to exert this receptor function. Previous work has shown that ephrinAs interact on RGC axons in cis with the neurotrophin receptors p75NTR and TrkB. P75NTR then was shown to be necessary for the repulsion of ephrinA-expressing RGC axons from an EphA substrate and for the downregulation of axon branching. In turn, an interaction of ephrinAs with TrkB as well as an increase in axonal ephrinA expression augments the axon branch-promoting activity of TrkB.

We now show that ephrinA6 is the necessary ephrinA component of the repulsive ephrinA/p75NTR receptor complex on chick RGC axons as axons lacking ephrinA6 no longer avoid an EphA matrix in stripe assay experiments. We also demonstrate that the branch-promoting activity of TrkB is dependent on ephrinA6 as a knockdown of ephrinA6 renders RGC axons insensitive to BDNF, the high affinity ligand for TrkB.

In sum our data further strengthen the hypothesis that a fine-tuned interplay of ephrinAs with p75NTR and TrkB is important for the guidance and branching of RGC axons.

Introduction

The retinotectal projection is a well-suited model system to understand molecules and mechanisms controlling topographic mapping. In this system retinal ganglion cell (RGC) axons from the temporal retina project onto the anterior tectum, while nasal RGCs project to the posterior tectum. In the perpendicular axis dorsal and ventral retina connect to lateral and medial tectum respectively. The retinotectal map develops in a number of steps involving activity-independent and activity-dependent processes. The initial ingrowth of retinal axons into the tectum in chick and mammals proceeds via an initial overshooting and subsequent topographically specific axon branching (Huberman et al., 2008, Lemke and Reber, 2005, O'Leary and McLaughlin, 2005).

The key players in retinotectal map development are members of the Eph family of receptor tyrosine kinases and the ephrins. A characteristic of this family—which is made up of the EphA and an EphB subfamilies—is their capacity to signal bi-directionally that is both Ephs and ephrins can function in a context dependent manner as either receptors or ligands (Klein, 2009). This potential is also realised for the retinotectal projection, where ephrinAs, for example, function as receptors when expressed in the retina, and as ligands when expressed in the tectum (Rashid et al., 2005). Overall multiple complementary EphA/ephrinA gradients control the steering of retinal axons to their correct location in the tectum along the antero-posterior axis. Thus there are gradients of EphA “receptors” in the retina (temporal > nasal) and ephrinA “ligands” in the tectum/SC (anterior < posterior), and of ephrinA “receptors” in the retina (nasal > temporal) and EphA “ligands” in the tectum (anterior > posterior). The idea has been put forward that the balance between signalling from these two gradient systems determines the location of topographically-specific branching of retinal axons (Marler et al., 2008, McLaughlin and O'Leary, 2005).

So far a functional characterisation has mostly focussed on ephrinA2 and ephrinA5. Nonetheless, in chick an additional ephrinA, ephrinA6, has been identified (Menzel et al., 2001). EphrinA6 shows clear differences in its amino acid sequence and affinity for EphA receptors when compared to ephrinA2/5, suggesting that ephrinA6 might have role/s divergent from those of ephrinA2/5. EphrinA6 is strongly expressed in the retina but only weakly in the tectum (Menzel et al., 2001) which is opposite to that of ephrinA2 and ephrinA5 which are more strongly expressed in the tectum than the retina (Menzel et al., 2001). Moreover, ephrinA6 is expressed not only in the RGC layer but also in all other layers, suggesting an involvement in establishing and/or stabilising intra-retinal organisation, while ephrinA2 and ephrinA5 are mostly confined to the RGC layer (Hornberger et al., 1999).

The function of ephrinA6 has been investigated so far only with regard to its tectal expression that is in its ligand function. Menzel et al. (2001) showed that ephrinA6 exerts a growth cone collapse inducing activity which is stronger for temporal than nasal axons, which correlates with the higher expression of EphA receptors on temporal than nasal RGC axons.

The aim of this investigation was to study the function of ephrinA6 on RGC axons. Previous stripe assay experiments have shown that RGC axons avoid an EphA substrate indicating that ephrinAs on RGCs can function as receptors and exert a repellent axon guidance function (Rashid et al., 2005). However, ephrinAs are GPI-anchored molecules (Davy et al., 1999, Huai and Drescher, 2001) and require therefore a transmembrane co-receptor/s to fulfil this receptor function. For this recently the neurotrophin receptor p75NTR was identified (Lim et al., 2008). Thus the repulsion of RGC axons from an EphA substrate (such as in stripe assays with alternating EphA7-Fc versus Fc lanes) is abolished using retinae from p75NTR mutant mice (Lim et al., 2008). The ephrinAs involved here were so far only little investigated (Rashid et al., 2005).

In addition, ephrinAs interact on RGCs also with other molecules, and modulate the function of cis-expressed EphA receptors (Carvalho et al., 2006) and TrkB receptors (Marler et al., 2008). TrkB contributes to retinotectal mapping by controlling retinal axon branching (Cohen-Cory and Fraser, 1995). An increase in the expression of ephrinAs on RGC axons further promotes TrkB signalling leading to an enhanced axon branching and synaptogenesis, suggesting that the level of ephrinA expression is important for TrkB function (Marler et al., 2008).

Our data presented here suggest that ephrinA6 is prominently involved in both guidance and branching functions of retinal axons, that is ephrinA6 is necessary for p75NTR -dependent repellent axon guidance, and for TrkB-mediated branching as knockdown of ephrinA6 leads to an abolishment of axon guidance and a substantial downregulation of axon branching.

Section snippets

Characterisation of RNAis mediating a knockdown of chick ephrinA6

EphrinA6 is the most prominently expressed ephrinA in the chick retina showing a high-nasal-to-low-temporal gradient. It is expressed in the ganglion cell layer as well as in other retinal layers (Menzel et al., 2001).

In order to address the function of ephrinA6, we sought to generate RNAis which efficiently knock-down ephrinA6. Using suitable software, a set of sequences from the ephrinA6 cDNA were selected and cloned into a U6 promoter based RNAi expression vector (Das et al., 2006). This

Discussion

During development of the retinotectal projection, multiple EphAs and ephrinAs with complex expression patterns in the retina and tectum are involved in targeting RGC axons to their correct termination zones in the tectum/SC. Our analysis shows that expression of ephrinA6 on chick retinal ganglion cell axons plays a key role in repellent axon guidance and interstitial branching.

Reagents and antibodies

BDNF was purchased from Promega. Anti-FLAG antibody was obtained from Sigma, and α-tubulin was from Sigma. The secondary antibodies, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were obtained from GE Healthcare. Laminin and all tissue culture reagents were from Invitrogen. Merosin was from Millipore. Recombinant EphA7-Fc chimera was obtained from R&D Systems and Fc control was from Calbiochem.

Cloning of chick ephrinA2 and -A6 for expression studies

Chick ephrinA2 and -A6 cDNAs were obtained by PCR and cloned into a CMV promoter containing

Acknowledgments

This work was supported by grants from the Wellcome Trust and the BBSRC. We thank Sarah Guthrie and Philip Gordon-Weeks for valuable comments on the manuscript.

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    Present address: Centre for Neuroendocrinology, Royal Free Campus, University College London, Rowland Hill Street, London NW3 2PF, UK.

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