Fig. 4

Characterization of a lamprey hoxα2 neural crest (NC)/hindbrain enhancer. a The mouse Hoxa2-Hoxa3 genomic region and its equivalent from the lamprey hoxα cluster are shown, with Hox gene exons annotated (blue arrows). hoxα2 upstream regions assayed for reporter activity in this study, with or without the c-Fos minimal promoter, are shown. b–e Lateral views (b, d) and frontal sections (c, e) of st24.5 lamprey embryos, comparing endogenous expression of hoxα2 (b, c) to GFP reporter expression mediated by hoxα2 −4 kb (d, e). Pharyngeal arches are numbered and rhombomeric expression detailed. Arrowheads point to PA2 NC expression. f Multiple sequence alignment of the Hoxa2 NC enhancer from gnathostomes with the lamprey hoxα2 enhancer, showing conserved sites (yellow). The positions of characterized mouse cis-elements (Krox20, Sox, RE2-3, NC3) are marked above the alignment. The enhancer schematic (a) shows the position of these elements within the assayed hoxα2 upstream regions, with conserved (shaded boxes) or divergent (empty boxes) cis-elements highlighted. Consensus binding motifs from the JASPAR database⁷⁶ for Krox20⁷⁷, Sox11⁷⁸, Meis1⁷⁹, and Pbx-Hox⁸⁰ factors are shown below the alignment, as well as sequences deleted in hoxα2 −4 kb ΔKrox20 and ΔNC3 variants. The non-aligning interval between these conserved regions is ~250–400 bp and varies in length between species. Supplementary Figure 5 contains the full alignment. g–j Lateral (g-i) and dorsal (j) views of st24.5 lamprey embryos showing GFP reporter expression driven by the enhancers detailed in a. Pharyngeal arches are numbered, with expression in rhombomeres (r) and somites (s) annotated. 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 Table 2 provides the number of embryos and details of specific expression for all constructs in lamprey