Tectal EphA3 guides nasal retinal ganglion cells axons during retinotectal mapping by competing with axonal EphA4 for axonal ephrin-As

FIORE, LUCIANO; CAVALLERO, GUSTAVO; MEDORI, MARA; DI NAPOLI, JENNIFER; DI SIERVI, NICOLÁS; DIAZ CARRIZO, JOAQUIN; RAPACIOLI, MELINA; SANCHEZ, VIVIANA; CARRI, NÉSTOR GABRIEL; SCICOLONE, GABRIEL

Oviedo

Congreso; SENC: 15 Congreso Nacional de la Sociedad Española de Neurociencia; 2013

Sociedad Española de Neurociencia

Tectal EphA3 guides nasal retinal ganglion cells axons during retinotectal mapping by competing with axonal EphA4 for axonal ephrins-As.  L. Fiore 1, G. J. Cavallero1, M. Medori1, J. I. Di Napoli1, N. Di Siervi1, J. Diaz Carrizo1, M. Rapaciolli2 , V. Sanchez1, N. G. Carri3 , G. Scicolone1 1Instituto de Biología Celular y Neurociencias ?Prof.Eduardo De Robertis? (IBCN-UBA-CONICET). Facultad de Medicina. Universidad de Buenos Aires, Argentina. 2Grupo Interdisciplinario de Biología Teórica, Universidad Favaloro. Buenos Aires, Argentina. 3Instituto Multidisciplinario de Biología Celular, (IMBICE-CIC-CONICET). La Plata, Argentina. Eph and ephrins are expressed in complementary gradients in both, the retina and the tectum and guide retinotectal projections by producing bidirectional signaling. Previously we demonstrated that tectal EphA3, acting like a second mapping force, stimulates axon growth of nasal retinal ganglion cells (RGC) toward the caudal tectum preventing them from branching in the rostral tectum (Ortalli et.al. 2012). The aim of this work was to study the molecular pathway which mediates the EphA3 action. We postulated that activation of axonal EphA4 decreases axon growth and that tectal EphA3 increases axon growth by reducing EphA4 activation throughout competing with axonal EphA4 for axonal ephrin-As binding. We used cultures of retinal explants from chicken embryos. Some of them were treated with EphA3-Fc or Fc, PIPLC (sheds ephrin-As), Lithocholic acid or KYL (inhibits ephrin-A-mediated EphA4 activation) and others were electroporated with EphA4, KiEphA4 (kinase-inactive dominant negative EphA4) or GFP pMES expression vectors. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrins-As. 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. e) Overexpression of EphA4 produces neurons with shorter axons than the neurons which express GFP or KiEphA4. Neurons expressing KiEphA4 have longer axons than the control. 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.