The neural crest gives rise to a large number of cell types in the vertebrate peripheral nervous system, including all autonomic and most peripheral sensory neurons. The mechanisms that generate this cell diversity e largely unknown. environmental factors, in the form of soluble and substrate-bound molecules, can affect neural crest differentiation, however, the absence of diagnostic tools has hampered the ability to determine their effects on the differentiation of specific neuronal types We propose to study the environmental regulation of neural crest differentiation using a novel murine culture system that we have developed. This culture system supports the generation of large numbers of neurons from neural crest cells. We will determine the phenotype of these neurons with a battery of tests that examine physiological and chemical properties. We will use specific pharmacological agents to determine the calcium channel repertoires of these neurons, measure their sensitivity to capsaicin and characterize their neurotransmitter content. In a series of parallel experiments we will examine the same properties in cultures of embryonic and neonatal mouse neurons. A direct comparison between authentic peripheral neurons and the neurons in crest cultures will allow us to identify how the environment is affecting neuronal differentiation. We will initially concentrate our studies on the effects of two cytokines, human leukemia inhibitory actor (hLIF) and mouse LIF (mLIF). Although these molecules share a high degree of homology, they have strikingly different effects on neural crest differentiation, with hLIF producing sympathetic-like neurons and mLIF producing sensory-like neurons. Our proposed studies will examine the effects of these two factors on the neuronal and neuronal progenitor cell survival and proliferation. In addition, we will examine sensory and sympathetic neurons that have differentiation, in vivo, in the absence of LIF. These studies will use neurons from transgenic mice that lack LIF expression. We will use the same battery of tests on these cells that are used o classify wild type neurons and neurons in crest cultures. Understanding the regulation of neural crest differentiation has broad implications for basic and biomedical research. A fundamental problem in developmental neurobiology is the elucidation of the mechanisms of neural indication and the regulation of neuronal phenotype. In addition, the abnormal development of the neural crest can have lethal or severely debilitating effects. These include malformations, deformations, disruptions and dysplasias. Research into the mechanisms that regulate the development of the neural crest will provide valuable insight into the potential causes of these abnormalities.