The long term goal of this project is to understand the steps in the development and maturation of the motor neuronal phenotype, and how extrinsic factors influence this process. Toward this end, our current efforts have been focused on generating a panel of monoclonal antibodies with which to identify somatic motor neurons during rodent development. Once the sequential appearance of such markers, during development, is determined, the antigenic profile of a motor neuron will serve as an accurate means of staging its state of differentiation. We have now generated several antibodies that selectively recognize somatic motor neurons. One of these shows a very strict specificity for this class of neurons, and immunoreactivity appears late in development. In contrast, two other antibodies stain relatively immature motor neurons. These results demonstrate that molecular markers of motor neuronal differentiation exists, and that monoclonal antibody techniques can be successfully used to produce probes to such antigens. We will continue to expand our panel of markers for this class of neurons, using novel immunization protocols, in order to obtain a complete antigenic profile of motor neurons throughout development. With the antibodies that we have already generated, our first aim is to examine carefully the spatial and temporal distribution of immunoreactive epitopes, in different populations of somatic motor neurons in the developing brainstem and spinal cord. We will then perturb afferent inputs or target contact to ask if acquisition or maintenance of immunoreactivity is dependent on these parameters. We will also biochemically characterize and identify the immunoreactive molecules present in motor neurons. Finally, we propose to examine the differentiation of motor neurons in vitro using a spinal slice preparation. We will ask whether the antigens expressed by motor neurons in situ can be acquired in culture, and with the same specificity and time course. These experiments, in conjunction with studies in situ, will provide information on the molecular differentiation of this important class of cholinergic neurons.