Evolution of vertebrates is intimately linked to the advent of the neural crest, a migratory and multipotent cell population that gives rise to many defining vertebrate characteristics, including a well-defined head and peripheral ganglia. Jawless fish (including the lamprey Petromyzon marinus) have bona fide neural crest cells, but do not have the full complement of neural crest derivatives, lacking jaws, sympathetic and possibly enteric ganglia. Here, we will test the hypothesis that the novel deployment of transcription factors in premigratory neural crest cells may have conferred new developmental potential onto this cell population, potentiating evolution of selected neural crest derivatives. Consistent with this hypothesis, two transcription factors, Twist and Ets1, are expressed in premigratory neural crest cells of gnathostomes but not lampreys. To test regulatory connections that may have facilitated production of novel vertebrate cell types, we will utilize RNA-seq together with novel transgenesis approaches to perform the following aims: Aim 1: Transcriptome analysis of lamprey premigratory cranial, vagal and trunk neural crest: To investigate the differences observed between cyclostome and gnathostome neural crest, we will perform RNA-seq analysis of cranial, post-otic (i.e. vagal) and trunk dorsal neural tubes containing premigratory neural crest cells from embryonic lamprey. We will confirm expression patterns of differentially expressed genes by in situ hybridization in lamprey and chick to find differentially expressed between agnathans and gnathostomes. Aim 2: Phylogenomic and functional analysis/dissection of conserved regulatory elements mediating expression of neural crest genes of lamprey and other vertebrates: We will identify regulatory regions of lamprey neural crest genes, initially focusing on Ets1 and Phox2b since they are differentially expressed between lamprey and gnathostomes. These will be tested by transgenesis in both lamprey and chick embryos. Aim 3: Utilize conserved regulatory elements to misexpress gnathostome neural crest genes in lamprey: We will utilize cross-species and/or endogenous enhancers to ectopically express missing transcription factors in lamprey to see if they promote formation of cell types that lamprey lack.