The long term-objective of this proposal is to gain a better understanding of the early specification steps that occur during human neural development. Understanding the early signaling events during this process is important both to expand our basic knowledge and to harness the full potential of human pluripotent cell lines for future nervous system therapies. While significant progress has been made in understanding these events in rodent models, relatively little has been done in human, largely due to lack of tissue availability. Human embryonic stem cells (hESCs) offer an accessible and manipulatable cell platform to model early neural development. It is thought that for many organisms, after neural induction, anterior neurectoderm (AN) is the default central nervous system (CNS) region generated, which is then patterned to posterior neurectoderm (PN) fates by posteriorizing signals. Recently, it has been shown that neural induction of human cells also leads to AN fates. However, patterning of hESCs to posterior fates is inefficient and after transplantation of these cultures into rodent brains, proliferative tumor overgrowths are frequently observed, indicating incomplete differentiation. This points out the need for basic studies of the mechanism of human neural induction and specification to improve the efficiency of generating desired neural cell subtypes. Classic studies in frog and mouse have shown that AN is specified via WNT antagonism by proteins such as dickkopf1 (DKK1). Using a novel, defined neural induction paradigm, our preliminary data indicate that AN commitment in hESCs is accompanied by marked increases in the WNT antagonists DKK1 and FRZB, indicating an evolutionary conserved mechanism. Here we propose to test the role of WNT signaling and antagonism in human neural specification, examining production of anterior and posterior fates as well as neural tumor formation. To do this we will test the following aims: 1. Determine whether WNT signaling is key for human AN specification. 2. Determine whether DKK1 and FRZB are endogenous determinants of human AN specification. 3. Determine whether reducing AN specification improves the efficiency of posterior fate specification and reduces tumor formation. PUBLIC HEALTH RELEVANCE: The results of the this study will provide critical information on how to generate nervous system cells from human embryonic stem cells in order to better model human nervous system disease and generate novel cells for replacement strategies.