Fig. S1

GFP reporter expression in NC mediated by variants of the zebrafish crestin promoter/enhancer in transient transgenic lamprey embryos. a, Versions of the zebrafish crestin promoter/enhancer tested for activity in zebrafish and lamprey embryos in this study. In zebrafish, the NC-specific activity of the crestin element depends upon consensus transcription factor binding sites for multiple transcription factors that are known to be part of a core NCGRN, including Sox10, Tfap2α and cMyc. Variants of the core minimal promoter/enhancer (crestin 296bp) were generated with mutations in these sites(ΔSox10, ΔTfap2α, ΔMyc). Regulatory elements were cloned upstream of the mouse c-Fos promoter. b-c, Lateral (b) and dorsal (c) views of two different transient transgenic lamprey embryos exhibiting GFP expression in NC (arrowheads) under the control of the crestin 1kb promoter/enhancer. Expression is first seen in the dorsal neural tube in NC cells as they start to delaminate, which then migrate to populate the pharyngeal arches at later stages. Even though the crestin element is specific to zebrafish and is not present in other gnathostomes, its cis-regulatory activity is conserved between lamprey and zebrafish. These data suggest that upstream regulatory factors that mediate activity of the crestin element in zebrafish may also be present in lamprey NC. d, Lateral views of st24 lamprey embryos injected with variants of the crestin promoter/enhancer. A minimal crestin promoter/enhancer is active in the lamprey NC; we found that the activity of this element in lamprey is also sensitive to perturbation of the same binding sites critical for activity in zebrafish. These results, coupled with the endogenous expression of SoxE1-3, Tfap2, and n-Myc in lamprey, suggest that the crestin element is interpreted in lamprey by components of an ancestral NC GRN that includes Sox, Tfap2α and Myc factors. GFP-expressing embryos shown are representative of the expression potential of the reporter construct in each case, as inferred from screening many (typically more than 100) injected embryos. Supplementary Tables 1-2 provide the injection statistics for crestin constructs in zebrafish and lamprey embryos.