Embryonic time course showing expression of hox genes in the lamprey hindbrain and cranial neural crest (NC). a Genomic organization of Hox genes in lamprey and mouse. Boxes represent Hox genes, which are organized into paralogue groups based on their sequence. The arrow above the clusters denotes the direction of Hox gene transcription. Lamprey hox genes from paralogue groups 1–3 were examined for NC expression in this study and their expression in cranial NC is denoted by green/white shading. b Lateral views of lamprey embryos from stages (st)21 to 26, showing hox gene expression domains in the developing head. Pharyngeal arches are numbered and rhombomere-specific domains (r) indicated. The arrowhead marks weak hoxδ2 expression in mandibular mesoderm at st26. c Frontal sections through lamprey embryos showing hox gene expression domains within the developing pharynx. Pharyngeal arches are numbered. d Schematic of a frontal section through the lamprey st24.5 embryonic pharynx with tissue domains annotated; NC domains are shaded in blue. Scale bars: 200 μm (b); 100 µm (c). e Schematic depicting hox expression in the lamprey hindbrain and NC at st23 and st24. ec, ectoderm; en, endoderm; m, mesoderm; mo, mouth; nc, neural crest; r, rhombomere; st, stage

Conserved activity of gnathostome Hoxa2 neural crest (NC) enhancers in zebrafish and lamprey. a Sequence alignment of gnathostome Hoxa2-Hoxa3 and lamprey hox2-hox3 gene loci against the human locus. Conserved non-coding sequences (pink), untranslated regions (UTRs) (cyan) and coding sequences (blue) are highlighted. The relative locations of the mouse hindbrain and NC cis-elements (top) are shown. Gnathostome Hoxa2 enhancers used for cross-species reporter analysis are detailed below the alignment. Letters within parenthesis indicate species of origin of the enhancer: zf, zebrafish; f, fugu; m, mouse. b, c Green fluorescent protein (GFP) reporter expression in zebrafish and lamprey embryos (lateral views), mediated by wild-type (b) and mutated (c) gnathostome NC enhancers. For zebrafish, the otic vesicle is circled and GFP expression in rhombomeres (r) and pharyngeal arches (2–5) indicated. Lamprey pharyngeal arches are labelled (2–4). 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. Injection statistics for the transient transgenic zebrafish embryos shown in c are given in Supplementary Table 3. d Frontal sections through the transient transgenic lamprey embryos shown in Fig. 3b, with GFP transcripts detected by in situ hybridisation, revealing expression in NC-derived mesenchyme (arrowheads) in the pharyngeal arches (numbered). Scale bars: 100 µm

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

Endogenous expression of meisC in neural crest (NC) overlaps with that of hoxα2 in lamprey embryos. a Lateral views (a, c) and frontal sections (b, d) are shown for embryos at st23 (a, b) and st24.5 (c, d). White lines in a and c denote planes of sections in b and d. Pharyngeal arches are numbered, arrows denote expression in NC. Scale bars: 200 μm (a, c); 100 µm (b, d). mb, mid-brain

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.

GFP reporter expression driven by gnathostome Hoxa2 NC enhancers in transgenic zebrafish reporter lines. Three independent lines are shown for each of the three gnathostome Hoxa2 enhancers. Letters in parenthesis indicate species of origin of the enhancer: zf, zebrafish; f, fugu; m, mouse. Lateral (top) and dorsal (middle) views are shown. Arrowheads denote GFP expression in PA2. Schematics at the bottom depict dorsal views of the rhombomeres (r) and pharyngeal arches (numbered), illustrating the consistent domains of activity observed between separate transgenic lines for each enhancer (blue shading). Abbreviations: HB, hindbrain; NC, neural crest; OV, otic vesicle.

Mutation of sites within gnathostome Hox2 enhancers and their influence on tissue-specific activity in transient transgenic zebrafish and lamprey embryos. a, Alignments of the NC3 region of the wild-type mouse (m) Hoxa2 NC enhancer with two variants, ΔNC3_1 and ΔNC3_2, showing the 15bp portions deleted in each variant. The positions of characterised hindbrain (purple) and NC (green) cis-elements are shown above the alignments. b, Dorsal views of transient transgenic zebrafish embryos with GFP expression mediated by the wild-type Hoxa2(m) enhancer and the two ΔNC3 variants. The left otic vesicle of each embryo is circled, with GFP expression in rhombomeres (r) and neural crest (nc) annotated. Embryos are at approximately 30 hours post-fertilisation. c, Alignment of a portion of the wild-type zebrafish (zf) hoxb2a NC enhancer with a variant in which the Pbx-Hox site has been mutated (ΔPbx-Hox). d, st24 transient transgenic lamprey embryos injected with the wild-type hoxb2a(zf) (lateral view) and mutated hoxb2a(zf)ΔPbx-Hox (dorsal view) enhancers. hoxb2a(zf) mediates expression in the hindbrain and neural crest, posterior to PA1 (pharyngeal arches are numbered), while this activity is lost in hoxb2a(zf)ΔPbx-Hox. GFP-expressing embryos shown in b and d are representative of the expression potential of the reporter construct in each case, as inferred from screening many (typically more than 100) injected embryos. The injection statistics for Hoxa2(m) and mutated variants in transient transgenic zebrafish embryos are provided in Supplementary Table 3, while those for the hoxb2a(zf) constructs in lamprey embryos are given in Supplementary Table 2.

GFP reporter expression driven by lamprey hoxα2 upstream regions in transient transgenic lamprey embryos. a, Lateral views of st24-25 transient transgenic lamprey embryos showing GFP expression in rhombomeres (r), somites (s) and NC of the pharyngeal arches (numbered), driven by the hoxα2 -4kb enhancer with or without the mouse c-Fos minimal promoter. In cloning the c-Fos promoter between the lamprey enhancer and the GFP coding sequence, two alternative reporter constructs were generated: hoxα2 -4kb cFosV1 with the upstream lamprey sequence fully intact, hoxα2 -4kb cFosV2 with the 5’UTR partially removed. In both cases, the insertion of the c-Fos promoter increased levels of reporter expression but did not influence tissue-specific expression domains. 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 (see Supplementary Table 2 for expression statistics). b, Frontal section through a transient transgenic lamprey embryo at st24.5, revealing GFP transcripts in the NC-derived mesenchyme (arrows) of the pharyngeal arches (numbered), driven by hoxα2 -4kb. c, Frontal section through a st24.5 lamprey embryo showing endogenous hoxα2 expression. Arrows indicate elevated expression in the NC-derived mesenchyme. The pharyngeal arches are numbered. Scale bars: 100μm. Abbreviations: r, rhombomere; s, somites.

Background GFP expression driven by the empty HLC vector in zebrafish and lamprey embryos. a, Lateral views of 30hpf/prim-16 stage transient transgenic zebrafish embryos injected with the empty HLC vector using Tol2-mediated transgenesis, showing low-intensity mosaic GFP expression in multiple tissue types including neurons and muscle cells. The otic vesicle is circled. b, Lateral views of st23.5 and st24.5 transient transgenic lamprey embryos injected with the empty HLC vector using I-SceI-mediated transgenesis, showing mosaic GFP expression in the yolk (y) and ectoderm, as well as cells lying dorsal to the yolk (arrowhead). 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.