For the nervous system to function properly, it is crucial that embryonic neurons acquire appropriate identities so they can develop the properties necessary for their later functions. The longterm goal of our laboratory is to understand the mechanisms underlying these processes. These mechanisms will be investigated by studying development of individually identified primary motoneurons in embryonic zebrafish. Our hypothesis is that the identities of primary motoneurons are specified by a series of signals that regulate expression of transcription factors that control neuronal identity and later features of development, such as axonal pathfinding. A series of experiments to test aspects of this hypothesis are proposed. Zebrafish primary motoneurons initially express a specific transcription factor, islet1, which is then downregulated; later islet1 or a related gene, islet2 is expressed in each specific primary motoneuron. The roles of the distinct phases of islet gene expression in motoneuron identity and axonal pathfinding will be tested using mutants and morpholino antisense oligonucleotides to knock down gene function. Retinoic acid, a well-known teratogen that can have devastating effects on human embryos, is synthesized in zebrafish during the time that primary motoneurons are being specified. Exposure to exogenous retinoic acid alters motoneuron numbers and patterning. The role of endogenous retinoic acid in motoneuron specification, and its interactions with other signals including the Hedgehog and Delta/Notch pathways, will be tested by a series of genetic gain- and loss-of-function experiments. The effects of retinoic acid on regulation of somite-derived signals that affect motoneuron identity will also be examined; potential signals will be identified using differential molecular screens. Interactions between specific primary motoneurons and the muscle fibers they innervate regulate their identities and survival. The role of the Delta/Notch signaling pathway in this process will be tested using a genetic approach. Although many genes regulating motoneuron development are known, many remain undiscovered. A mutagenesis screen will be undertaken to identify new genes involved in specifying motoneuron identities.