Expression of ephrinA5 during development and potential involvement in the guidance of the mesostriatal pathway

Abstract

Identifying guidance cues that direct axon growth to their final connections during development is of crucial interest if we aim to repair circuits damaged in adulthood following neurodegenerative disorders or common traumatic injuries. In this work, we set out to determine the ephrinA5 guidance molecule involvement in the establishment of the mouse mesostriatal pathway during development. We showed, in vitro and in vivo, that a proportion of mesencephalic dopaminergic cells express the ephrinA5 receptor, EphA5. Moreover, we observed, using stripe assays, that ephrinA5 purified protein has a repulsive effect on most of the mesencephalic dopaminergic projections. In vivo, we detected rostro-caudal and ventro-dorsal ephrinA5 protein expression gradients in the vicinity of the dopaminergic axons in the ventral telencephalon and in the striatum, during the embryonic and early postnatal development. In addition, other EphA5 ligands were also detected in the mesostriatal pathway. Together, these expression patterns suggest that, ephrinAs and more specifically ephrinA5, may be actors in the guidance of dopaminergic projections. Further studies will focus on identifying the molecular specificity of these guidance cues, taking into account the mesencephalic dopaminergic heterogeneous neuronal population. This may help increase the integration of neuronal transplants in the mature lesioned brain or provide tools to re-establish mesostriatal circuits in vivo.

Introduction

Axonal growth and guidance resulting in the establishment of neuron connections in the mammalian nervous system mainly occur during embryogenesis when outgrowing axons have to travel long distances to reach their target neurons. Along their pathway, they are directed by attractive, repulsive, and adhesive guidance cues present in their local environment. Some of these are part of the extracellular matrix, others are secreted in the environment leading to a long-distance guidance or are cell membrane-anchored (Plachez and Richards, 2005, Dickson, 2002, Chilton, 2006, Yamamoto et al., 2003). The identity and functions of these molecules have been well documented particularly for the retinotectal and the thalamocortical systems (Knöll and Drescher, 2002, Haupt and Huber, 2008, Maroof and Anderson, 2006).

However, in the mesostriatal dopaminergic system, the identity of chemoattractive and chemorepulsive signals guiding the dopaminergic axons to the striatum is still unclear. This system establishes itself during early embryogenesis: dopaminergic neuron somas located in two mesencephalic adjacent regions, the substantia nigra (SN) and the ventral tegmental area (VTA), send out projections forming the medial forebrain bundle (MFB) that connect to the dorsal and the ventral striatum (Gates et al., 2004). During their travel to the striatum these dopaminergic projections cross diencephalic and telencephalic regions that are thought to produce local cues that guide them to their final target (Nakamura et al., 2000). Gates et al. (2004) have localized chemoattractive and chemorepulsive regions that may direct the nigro-striatal pathway through intermediate targets before its connection to the striatum. They showed that the MFB and the striatum have an attractive influence on mesencephalic axons expressing tyrosine hydroxylase (TH+ axons), while brain stem and cortical regions have an inhibitory effect. In return, the thalamus did not seem to have any influence on the guidance of these projections. Several conserved families of guidance molecules are thought to be responsible for these attractive, repulsive or inhibitory effects. Proteoglycans have a permissive or inhibitory effect on TH+ embryonic mesencephalic projections in vitro (Macé et al., 2002). Netrin and slit have an attractive and repulsive influence respectively, on mesencephalic dopaminergic projections in vitro (Lin and Isacson, 2006). In vivo, several studies have detected the expression of chemokines in the nigro-striatal system (Banisadr et al., 2003). A more recent study showed attractive and repulsive roles for semaphorin3C and semaphorin3F respectively expressed in the pretectum, whereas semaphorin3A expressed in the striatum was shown to enhance axon growth (Hernandez-Montiel et al., 2008).

In addition, several ephrin guidance molecules have been found to be expressed and involved in the mesostriatal pathway formation. Ephrins (As and Bs) constitute a large family of membrane-anchored guidance molecules that participate in the refinement of the neuronal connections by cell-to-cell contact. Their action involves an Eph–ephrin (receptor–ligand) interaction inducing attraction or repulsion, adhesion or de-adhesion, and migration (Egea and Klein, 2007). Nine ligands (ephrinA1–A6 and ephrinB1–B3) and sixteen receptors (EphA1–A10, EphB1–B6) have been identified to date. EphrinB2 is expressed in the ventral striatum, and EphB1 is expressed in both striatum and the substantia nigra compacta (SNc) (Richards et al., 2007, Yue et al., 1999). SNc dopaminergic projections expressing EphB1 receptor are thought to interact with ephrinB2 ligand expressed in the ventral striatum, which might prevent these dopaminergic projections to connect to this structure.

Several EphAs–ephrinAs have also been detected in this system. EphrinsA1, A2, A3, A4, A5 and EphA4, A7 are expressed in the striatum (Janis et al., 1999) whereas EphA5 was mainly found in the ventral mesencephalon (Yue et al., 1999). EphrinAs–EphAs may play an important role in the establishment of the mesostriatal connections since disruption of EphA–ephrinA signaling in the nigro-striatal system reduces dopaminergic innervation to the striatum without changing the number of dopaminergic cells in the SN (Sieber et al., 2004). This suggests that less dopaminergic projections are able to reach the striatum in mice lacking EphA–ephrinA signaling. Overall, these studies are in favor of an involvement of ephrinAs in the mesostriatal pathway establishment. More specifically, EphA5 and ephrinA5, expressed in the ventral mesencephalon and in the striatum (Yue et al., 1999, Janis et al., 1999, Passante et al., 2008, Cooper et al., 2009a), may participate in the guidance of the mesostriatal axons.

In this study, we set out to identify the influence of ephrinA5 on dopaminergic mesencephalic axons in vitro, and to examine the expression of ephrinA5 protein along the mesostriatal pathway during development. For this, we used functional in vitro stripe assays, and in vivo ephrinA5 immunodetection on embryonic and newborn mouse brains. We showed that dopaminergic mesencephalic cells express the ephrinA5 receptor, EphA5, and are repulsed by ephrinA5-purified protein. Moreover, immunodetection of dopaminergic pathway along with the expression patterns of ephrinA5 in the developing mouse embryonic brain, suggested that ephrinA5 may play a role in the guidance of mesencephalic dopaminergic striatal projections. Finally, detection of other putative EphA5 ligands in the vicinity of the mesostriatal pathway and the absence of any alteration of this pathway in ephrinA5 knock-out mice, suggest that other ephrinAs, in addition to ephrinA5, may be involved in the establishment of this pathway.