A basic feature of the nervous system is the high degree of specificity with which synaptic connections among neurons and between neurons and muscles are formed. It is of fundamental importance to learn how this specificity arises. How does a nerve cell know when and where to grow and what to innervate? The proposed experiments will explore these questions 1) by determining the patterns of axonal growth and target selection by developing, identified vertebrate motoneurons; 2) by characterizing the macromolecular nature of the environment through which their axons grow; and 3) by studying the genetic basis of the differentiation and pathway selection of these neurons. The patterns of axonal growth and target selection will be determined by observing living motoneurons, in vivo, as their axons grow and make synaptic contacts wih muscle fibers. Primary motoneurons which are few in number and uniquely identifiable in zebrafish embryos will be labeled with fluorescent dyes and their axons will be followed as they grow in these rapidly developing, optically clear embryos. The specificity of synaptic connections will be assayed in older animals using physiological recording procedures. These data will, for the first time, enable us to provide a description of the """"""""dynamic"""""""" aspects of specific in situ neuronal development in vertebrates. The macromolecular nature of the environment through which the axons of these identified motoneurons grow will be determined using histochemical and immunocytochemical markers of some of the constituents of the extracellular matrix and cell surfaces. Additionally, new markers to as yet uncharacterized molecules along these pathways in zebrafish will be generated using monoclonal antibody procedures. The genetic basis of neurogenesis, connectivity and muscle development will be studied by inducing and analyzing mutations that affect specific aspects of the system's development. The novel technique of insertional mutagenesis will be used to introduce deficiencies into the zebrafish genome. The developmental consequences of these deficiencies will be maintained and analyzed using simple screens. The genes identified by these mutations will be automatically labeled by the insertion and, ultimately, will be isolated for further study.
