The neural crest is a uniquely vertebrate cell type that is thought to have played an important role in vertebrate evolution by forming peripheral ganglia and jaws, thus facilitating predation and expansion of the brain. We recently identified a “cranial-specific” neural crest transcriptional subcircuit in jawed vertebrates (gnathostomes) that imbues this neural crest population with the unique ability, absent from trunk neural crest, to form craniofacial cartilage that is critical for jaw formation. Here, we propose to examine whether homologous genes are expressed in the cranial premigratory neural crest of lamprey, a jawless basal vertebrate (agnathan). Our preliminary results suggest that many of these genes are “missing” from the lamprey's premigratory cranial neural crest, thus challenging the hypothesis that invention of neural crest in vertebrates gave rise to a “New Head”. Accordingly, we hypothesize that there was progressive expansion of neural crest derivatives during the course of vertebrate evolution. This likely occurred by addition of new enhancer elements into the premigratory neural crest that conferred novel developmental potential onto this cell population. As case in point, lamprey lack a vagal neural crest that in gnathostomes forms the enteric nervous system. To test this hypothesis, we will perform lineage analysis, analyze transcription factors and regulatory regions of selected genes across agnathan and gnathostomes. We will identify putative enhancers, dissect their regulatory inputs, and test the ability of these regions to drive reporter expression in lamprey as well as cross-species, in the zebrafish. The results promise to elucidate how new cell types arose during vertebrate evolution under the umbrella of the neural crest.