How a pluripotent cell becomes committed to a specific differentiation pathway is a central question in developmental biology. The overall objective is to understand such commitment decisions by studying the interstitial lineages of hydra. The multipotent stem cell of this system gives rise to four classes of differentiation products: neurons, nematocytes, secretory cells and gametes. In previous segments of this work an extensive body of information has been gained about the stem cells and the four classes of products at the cellular level. In this proposed segment, the focus will be on the commitment decisions that occur during neuron differentiation. Two are known. One is the restriction of the multipotent stem cell to neuron differentiation, and the other is a decision late in the pathway as to neuron phenotype. The aims here are three-fold. [1] To complete an understanding of the neuron lineage in order to determine if there are any more stages where a decision as to cell type is made. This will be done by marking individual interstitial cells and determining the differentiated cells they produce. [2] Neuron phenotype determination is position-dependent in hydra. For one subset of neurons this effect is mediated by the phosphatidylinositol pathway. The aim is to identify the signal that is responsible for the position-dependent effect, and determine if it is acting directly or indirectly on the neurons. Grafting experiments involving tissue with a variety of cell compositions coupled with diacylglycerol treatments will determine if the effect is direct or indirect. Examination of a number of peptides and proteins known to affect neuron behavior or be associated with neurons in hydra will determine if any is the signal. [3] To identify genes involved in the commitment decisions. The approach is to determine if specific genes involved in neuron differentiation decisions in both vertebrates and arthropods are present in hydra. These will be identified using PCR, and the isolation and sequencing of corresponding cDNAs. Expression patterns of the gene at the mRNA and protein level will determine if they affect the neuron lineage. The initial candidates will be members of the achaete-scute family of genes.