A key, but insufficiently understood area of neural development is the molecular basis of neurospecificity. At the physiological level, one of the best studied examples of synaptic specificity in vertebrates is the rostrocaudal, positional bias displayed by spinal cord axons as they innervate sympathetic ganglia or transplanted intercostal skeletal muscles. In an effort to examine the molecular basis of this positional specificity, we used the cyclophosphamide immunosuppression method to produce of monoclonal antibody (mAb), ROCA1, which binds preferentially to rostral ganglia and rostral intercostal nerves. ROCA1 finds specifically to a 6OkD and a 26kD protein. We have identified the former antigen as the neuron-specific, intermediate filament, peripherin, and immunoblots demonstrate that peripherin is expressed in a graded manner in intercostal nerves, declining in caudal segments. The 26kD antigen is a surface membrane protein expressed by neurons and glia. ROCA1 binding to the 26kD protein in sections of nerves and ganglia declines dramatically in caudal segments, apparently due to a masking of this epitope. The proposed research will extend these findings in four directions. (i) The 26kD protein has been purified for amino acid sequencing. This data will be used to isolate the corresponding cDNA clones. Cloning will allow comparison with sequences of known proteins and functional motifs, (ii) Results with likely anti-idiotype Abs suggest that the 26 and 6OkD proteins may interact with each other. This will be further explored using the anti-idiotype Abs and direct binding studies. (iii) The cell- and position-specific expression of both peripherin and the 26kD protein is being explored in rat embryos with several Abs. (iv) Expression of peripherin and the 26kD protein is being examined in cell culture. Questions under study include: is peripherin preferentially expressed by rostral neurons in culture and, if so, does that difference require association with glia; does the apparent masking of the ROCA1 epitope on the 26kD protein in caudal glia depend on association with caudal neurons.
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