There are three specific aims in the present proposal.
Aim 1 seeks to determine whether the complementary distribution of EphB2 and its ligand, ephrin B1 regulate dorso-ventral patterning in the retina. Ephrin B1 is in dorsal retina and EphB2 is in ventral retina. Studies include: (a) in vivo misexpression using RCAS virus, using ephrin-B1DC +/- EGFP and EphB2DC and examining retinas between E6 and E16. The investigator(s) will look for growth abnormalities and axon patterning using DiI labeling. (b) in vivo misexpression using replication incompetent virus (RSV based with lacZ and internal ribosome entry site (IRES) to misexpress Eph B2 in hostile ephrin B1 domain and vice versa to test the hypothesis of bidirectional signaling, and (c) in vitro assays with cultured retinal cells and transfected lines. The investigator has generated CHO cells transfected with EphB2 and will generate ephrin-B1 expressing cells. The investigator(s) will look for segregation between them and patching of receptors/ligands and will analyze both directional migration of retinal cells in transfilter assays and growth cone collapse.
Aim 2 : Eph A3 is expressed in posterior retina and ephrin A5 in anterior retina. The investigator will determine whether the complementary pattern regulates AP (anterior-posterior) patterning of visual structures. Studies include: (a) misexpression of EphA3 and ephrin A5 using replication incompetent viruses, (b) in vitro assays using CHO cells transfected with EphA3 or ephrin A5, and (c) examination of directional migration and axon collapse.
Aim 3 will determine whether the polarized tyrosine phosphorylation of EphA4 regulates patterning. Preliminary results show that EphA4 and B2 exhibit a polarized pattern of phosphorylation in retina. A4 expression is high dorsoanteriorly and low ventroposteriorly. Studies include (a) alteration of activation using a constitutively active myc-EphA3 lacking most of extracellular domain inserted into RCAS and myc-ephrin A5 and (b) examination of axon trajectories using DiI.
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