Regulation of axonal EphA4 tyrosine-phosphorylation mediates the effect of EphA3 on retinal ganglion cell axon growth during retinotectal mapping

FIORE, LUCIANO; DI NAPOLI JENNIFER; CASTRO, IGNACIO; BENÍTEZ ROCÍO; RAPACIOLI MELINA; CARRI NÉSTOR GABRIEL; SCICOLONE GABRIEL

Nueva Orleans

Congreso; Neuroscience 2012; 2012

Society for Neuroscience

Regulation of axonal EphA4 tyrosine-phosphorylation mediates the effect of EphA3 on retinal ganglion cell axon growth during retinotectal mapping. The retinotectal system is useful to study topographic map formation because nasal retinal ganglion cells (RGC) project to caudal tectum and temporal ones connect to rostral tectum. Eph receptor tyrosine kinases and their ligands, the ephrins, are expressed in complementary gradients in both, the retina and the tectum and guide retinotectal projections by producing bidirectional signaling. Ephrin-As located in the caudal tectum repel temporal axons by activating EphA3/A4. Ephrin-As expressed in nasal RGC activate EphA4 converting nasal RGC less sensitive to tectal ephrin-As. Previously we demonstrated that tectal EphA3, acting like a second mapping force, stimulates axon growth of nasal RGC toward the caudal tectum preventing them from branching in the rostral tectum (Ortalli et. al. 2012). It is not known which molecule/s mediates these effects of EphA3 ectodomain. Thus, the aim of this work was to study the molecular pathway which mediates the EphA3 action. In that way we postulated that EphA4 activation decreases axon growth and that tectal EphA3 increases axon growth by reducing EphA4 activation through competing with axonal EphA4 for axonal ephrin-As binding. Methods: We used cultures of nasal and temporal retinal explants from chicken embryos treated with EphA3-Fc or Fc (control), PIPLC (sheds ephrin-As) or KYL (inhibits ephrin-A-mediated EphA4 activation). We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrins-As. Results: We showed that: a) Nasal RGC axons present higher levels of ephrin-As, colocalization of ephrin-A2 and EphA4, and tyrosine-phosphorylated EphA4 than temporal RGC axons. b) Axonal response to EphA3 ectodomain is associated to ephrin-A expression and EphA4 tyrosine phosphorylation. c) The EphA3 ectodomain and ephrin-A shedding both decrease the degree of EphA4 Tyr-602 phosphorylation. d) Removal of axonal ephrin-As and inhibition of ephrin-A-mediated-EphA4 signaling recapitulate the effects of EphA3 ectodomain on RGC axon growth and branching. These data show that treatment with the EphA3, ephrin-A shedding and EphA4 inactivation all increase RGC axon growth, decrease axonal filopodia density and inhibit EphA4 tyrosine phosphorylation. Discussion: These results support the idea of a novel molecular mechanism whereby tectal EphA3 increases axon growth toward the caudal tectum and collaborate to inhibit axon branching in the rostral tectum by decreasing ephrin-A-mediated-EphA4 forward signaling. Founded by UBA and CONICET.