Both neural crest progenitors and ectodermal placode cells arise from the neural plate border (NPB). While the neural crest gives rise to the craniofacial skeleton, the placodes form the lens, ear and olfactory system; both contribute to cranial sensory ganglia. However, rather than being fixed to a neural crest or placodal fate, our results show that cells in the neural plate border appear to coexpress transcription factors characteristic of multiple lineages, ranging from neural crest to neural to placodal. Moreover, we find there is a stem cell niche within the dorsal neural tube that expresses pluripotency factors including Sall4, Nanog, Oct4, Klf4, and cMyc as confirmed in our single cell RNA-seq data. Thus, the question of what maintains stem cells with the potential to form neural crest, placode and neural tube derivatives at the neural plate border remains open. Our preliminary data suggest that Sall4 may form a feed-back loop with other multipotency genes, including Pou5f3/Oct4 and Sall1. Moreover, overexpression of Sall4 prevents neural crest specification and upregulates Oct4. To test the hypothesis that these pluripotency factors maintain multipotency of the neural plate border and that their downregulation is necessary for neural crest and/or placode specification, we will explore the effects of their gain and loss of function, identify enhancers that mediate their expression and examine dynamic changes in their gene expression during neural plate border maturation. To this end, the following aims will be performed. Aim 1: Effects of ectopic maintenance or loss of pluripotency factors on neural crest and placode development. We will test the role of pluripotency factors Sall4, Oct4, Nanog and Klf4 in vivo in neural plate border development using gain and loss of function approaches coupled with single cell RNA-seq. In particular, we will test the hypothesis that these pluripotency factors maintain the multipotency of the neural plate border and their downregulation is necessary for completion of neural crest specification. We will also examine other transcription factors to test their role in driving lineage specification at the neural plate border. Aim 2: Identification and cis-regulatory analysis of putative enhancers mediating expression of pluripotency factors and neural plate border transcriptional regulators. Detailed cis-regulatory analysis allows identification of active enhancers and their direct inputs, both positive and negative, thus informing upon gene regulatory network connections. We propose to use single cell ATAC-seq to identify putative regulatory elements active at the forming neural plate border, particularly those mediating expression of pluripotency factors and transcriptional regulators. Putative enhancers will be tested for inputs and their ability to drive expression. Aim 3: Dynamic analysis of enhancer-mediated expression during maturation of the neural plate border. By coupling live imaging with enhancer d...