Development of neuronal type specific cDNA libraries

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Since neuronal cells have own characteristics from the beginning of their differentiation, the correct path finding to their correct target cells is essential for establishing neural network. Selective axonal growth is considered to be achieved by active interactions between the cues provided along the pathway and the recognition molecules localized on the growth cones. To identify such cues and recognition molecules, we focused on the chick embryonic lumbar motoneurons, because their axonal growth is analyzed in great detail and we can obtain enough materials from the embryos. We tried to identify molecules that are uniquely expressed on motoneurons or their subtypes from the cDNA libraries made from the purified embryonic day 5 (E5) chick motoneurons by panning, using antibody against SC1 that is uniquely expressed on motoneuron cell surface. We then performed RT-PCR for receptor tyrosine kinases (RTKs) and mRNA differential display. To date, RTKs comprise at least 16 subfamilies based upon their structural motifs. From enriched motoneurons, we identified 12 RTK subfamilies and found by in situ hybridization that Cek8, a member of Eph subfamily, is specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles. Furthermore, Cek4 of the same family is expressed on motoneurons innervating axial muscles, whereas Ret RTK is expressed in all motoneurons. From the mRNA differential display we cloned nearly 40 cDNA clones and found at least 3 novel molecules are uniquely expressed on all or subpopulation of motoneurons. We are now analyzing the biological functions of Cek4, Cek8, Ret and the novel molecules expressed on the motoneurons.