The objective of this project is to understand the possible role that eph receptor tyrosine kinases (RPTK) may play during neuronal differentiation. The RN33B cell line differentiates in vivo after transplantation into the neonatal and adult rat brain. This cell line shows neurite outgrowth formation and a morphology typical of endogenous neurons when transplanted in the rat cerebral cortex or hippocampus. The high levels of eph RPTK ligand expression in the central nervous system, along with the expression of at least one eph RPTK ligand expression in the central nervous system, along with expression of at least one eph RTK in RN33B cells, has led us to hypothesize that this protein could mediate the morphogenesis observed in vivo. In addition, sever eph RPTKs have been implicated in axonal targeting, suggesting that this family of RPTKs are involved in the process of neural differentiation. To address the question, we will screen and sequence clones derived from RN##B cells using RT-PCR with degenerate oligonucleotides for the conserved regions of the eph RPTK. Once the type(s) of eph RPTK has been determined, developmental studies at the mRNA and protein level will be done with Northern blotting, Western blotting and immunohistochemistry techniques on RN33B cells in vitro. The possible role of eph RPTK in differentiation will be tested by the use of a dominant negative construct, to inactivate the RN33B cell eph RPTK, and analyze its effect on morphogenesis after transplanted into the neonatal rat brain. The analysis will make a significant contribution to the understanding of neuronal differentiation in vitro and in vivo, when a neural cell line is used as a model system to study differentiation in culture during neural grafting.
