Double role of EPHA3 in the axonal guidance of retinal ganglion cells.

ORTALLI AL; NG CARRI; G ALVAREZ; Y OTROS

Buenos Aires

Congreso; ISER; 2006

International Society for Eye Research

Many axons establish an orderly arrangement of connections in their target field, termed a topographic map. A widely studied example of topographic map is the retinotectal system, where nasal retinal ganglion cells (RGCs) connect to the caudal tectum and temporal RGCs contact the rostral tectum. The recognition of graded cues in the target is the main mechanism that participates in the map formation. Eph receptors and ephrins are expressed in complementary gradients in both organs. EphrinAs located in the caudal tectum repel temporal axons by activating the EphA3 and EphA4 on these axons. However, it is not known whether the EphA3 expressed in the rostral tectum participates in retinal axon guidance and how the axons of nasal RGC grow towards the caudal tectum. We tested the effects of the EphA3 ectodomain regarding axonal behavior. Retinal explants and dissociated retinal neurons from 6-days-old chick embryos were cultured on EphA3-Fc or Fc substrates. Our results demonstrate that EphA3 ectodomain promotes axon growth in nasal and temporal cells. This effect is dose dependent and differs between both populations of RGCs. In addition, we performed time-lapse experiments to examine the behavior of growth cones extending on 293 cells transfected with EphA3-GFP or with GFP (green fluorescent protein). The results show that growth cones expand upon contact with EphA3-expressing cells but not control cells. Furthermore, nasal growth cones extend over EphA3-expressing cells while temporal growth cones adhere instead of extending. These results demonstrate that EphA3 ectodomain stimulates axonal growth, suggesting that both axonal and tectal EphA3 might guide axonal growth over the tectal surface, acting as receptor of the repulsive tectal ephrinAs and as a stimulatory ligand, respectively. Since ephrinAs (GPI-anchored proteins) are expressed in optic fibers, we investigated whether axonal GPI-anchored proteins mediate the stimulating effect of EphA3 on axonal growth. Thus, retinal explants grown on EphA3-Fc or Fc were treated with increasing doses of PI-PLC, an enzyme that sheds GPI anchors. EphA3-Fc-stimulated nasal explants increase their axonal length at low PI-PLC doses, but reduce it at high doses. A weaker effect was observed in temporal explants. Control explants increase axonal length at every PI-PLC dose. The positive effect of PI-PLC on control axons suggests that GPI-anchor proteins could reduce axonal growth through axonal EphAs activation. The dual effect of the enzyme on EphA3-stimulated axons also suggests that the lack of ephrinAs and other GPI-anchored proteins impairs EphA3-elicited axonal growth. The weaker effect of PI-PLC on temporal axons suggests that the retinal ephrinAs, expressed in a decreasing nasotemporal gradient, could act as receptors of EphA3. This supports the idea that ephrinAs could have a double role as both repulsive tectal ligands for axonal EphAs and receptors of stimulatory tectal EphA3. Supported by grants from CONICET and UBA. Argentina. Keywords cell-cell communication, retinal development, retinal connections, networks, circuitry