Human Brachyury enhancer elements T3, C, and I are active in different species.

A Human Brachyury/T/TBXTB locus with surrounding gene loci adapted from UCSC genome browser. Repeats marked in black using the RepeatMasker track; additional tracks include the ENCODE conserved cis-regulatory elements (cCREs) and layered H3K27ac signals. Further annotated are approximate amplifications (light orange) and the minimally amplified region (dark orange) in chordoma tumors. ATAC-sequencing (light blue peaks) and T ChIP-sequencing (dark blue lines) suggest enhancer elements (light pink highlight, not active; light blue highlight, active) that are conserved in mouse and the marsupial Monodelphis domestica. B–D Representative F0 zebrafish embryos injected with the human enhancer elements hs_T3 (B), hs_C (C), and hs_I (D) showing mosaic mCerulean reporter expression in the notochord at 24 hpf and expression of ubi:mCherry as injection control. N represents the number of animals expressing mCerulean in the notochord relative to the total number of animals expressing mCherry. Scale bar in (B): 0.5 mm, applies to B, C. E–G Representative images of stable transgenic F2 embryos at 48 hpf for each of the human enhancer elements hs_T3, hs_C, and hs_I crossed to Tg(drl:mCherry) that labels lateral plate mesoderm and later cardiovascular lineages. Transgenic F2 embryos recapitulate the F0 expression pattern in the notochord, with hs_T3 (E) additionally expressing mCerulean in the pharyngeal arches and fin, and hs_I (G) in the proximal kidney close to the anal pore. Enhancer element hs_C (F) stable transgenic lines have lower relative notochord reporter activity than hs_T3 and hs_I. Scale bar in (E): 0.5 mm, applies to E–G. H–J Representative F0 axolotl embryos at peri-hatching stages expressing mCerulean from the human enhancers hs_T3 (G), hs_C (H), hs_I (I). N represent the number of animals expressing mCerulean in the notochord relative to the total number of animals showing any mCerulean expression. Scale bar in (H): 1 mm, applies to H–J. K, M, and O Representative images of transgenic E9.5 mouse embryos expressing lacZ (encoding beta-galactosidase) under the human enhancers hs_T3 (K), hs_C (M), and hs_I (O) visualized with X-gal whole-mount staining. While hs_C and hs_I express beta-galactosidase in the entire notochord, beta-galactosidase expression from hs_T3 is restricted to the posterior notochord. Black asterisk marks absence of beta-galactosidase in the anterior notochord. N represent the number of animals expressing beta-galactosidase in the notochord relative to the total number of animals with tandem integrations at H11. Dotted lines represent the sectioning plane. Scale bar in (K): 0.5 mm, applies to (K, M, O). L, N, P Representative images of Fast Red-stained cross sections from embryos shown on the left, hs_T3 (L), hs_C (N), and hs_I (P). Black arrowheads point at notochord, and inserts show notochords at 2x higher magnification. Scale bar in (L): 0.25 mm, applies to L, N, P. The species silhouettes were adapted from the PhyloPic database (www.phylopic.org).

Identified TBXT binding sites in the enhancer elements are essential for reporter activity.

A Sequence of the human TBXT binding site (T-box) using JASPAR. B FIMO output with location of the T-box, statistical significance, and matched sequence within the enhancer elements. P-values were calculated by FIMO which computes a log-likelihood ratio score for each position in the sequence, then converts this score to a P-value, and then applies false discovery rate analysis to estimate a Q-value for each position. C Schematic depiction of the wildtype human enhancer elements with the TBXT binding site/T-box (pink, red, purple boxes) and the enhancer elements without the respective T-box sites (ΔTbox). The human enhancer elements are depicted in the reverse complement direction. Tbox130-145, Tbox277-292, Tbox309-324: p < 0.00008, Tbox184-199: p < 0.005, Tbox201-216: p < 0.008. D–I Injection of the wildtype enhancer elements hs_T3 (D), hs_Cshort (F), and hs_I (H) as reporter constructs results in mApple fluorophore expression in the notochord at 48 hpf, whereas injection of hs_T3ΔTbox (E), hs_CshortΔTbox (G), and hs_IΔTbox (I) show complete loss of notochord expression (asterisks in E, G, I). Arrowheads (D–I) mark EGFP expression in the pineal gland from the transgenesis marker exorh:EGFP. Scale bar in (D): 0.5 mm, applies to D–I.

Mouse Brachyury enhancer elements are active in different species.

A Mouse Brachyury/T/TBXTB locus adapted from the UCSC genome browser. Repeats marked in black using the RepeatMasker track; additional tracks include the ENCODE cCREs, H3K27ac (yellow), H3K4me (red) and DNase (green) signals. ATAC-sequencing (light blue peaks) and T ChIP-sequencing (dark blue lines) indicate enhancer elements (light pink highlight, not active; light blue highlight, active) that are conserved in human and Monodelphis. B–D Representative F0 zebrafish embryos injected with the mouse enhancer elements mm_T3 (B), mm_C (C), and mm_I (D). mm_T3 and mm_I show mosaic mCerulean reporter expression in the notochord at 24 hpf and mosaic ubi:mCherry expression as injection control. Mouse enhancer element mm_C is not active in the zebrafish notochord (asterisk in C). N represent the number of animals expressing mCerulean in the notochord relative to the total number of animals expressing mCherry. Scale bar in (B): 0.5 mm, applies to (B–D). E, G, I Representative images of transgenic E9.5 mouse embryos expressing lacZ (encoding beta-galactosidase) under the mouse enhancer elements mm_T3 (E), mm_C (G) and mm_I (I) visualized with X-gal whole mount staining. While mm_T3 and mm_I express beta-galactosidase in the entire notochord, beta-galactosidase expression from mouse mm_C is absent (asterisk in G). N represent the number of animals expressing beta-galactosidase in the notochord relative to the total number of animals with tandem integrations at H11. Dotted lines represent the sectioning plane. Scale bar in (E): 0.5 mm, applies to E, G, I. F, H, J Representative images of Fast Red-stained cross sections from embryos shown on the left, mm_T3 (F), mm_C (H), and mm_I (J). Black arrowheads point at notochord, and inserts show notochords at 2x higher magnification. Scale bar in F: 0.25 mm, applies to F, H, J. The species silhouettes were adapted from the PhyloPic database (www.phylopic.org).

Monodelphis Brachyury enhancer elements are active in different species.

A Monodelphis Brachyury/T/TBXTB locus adapted from the UCSC genome browser. Repeats are marked in black using the RepeatMasker track. Further annotated are tracks containing N-SCAN gene predictions and 9 Species Conservation. The light blue highlighted boxes mark the Monodelphis enhancer elements T3, C and I and their conservation in other species. B–D Representative F0 zebrafish embryos injected with the Monodelphis enhancer elements md_T3 (B), md_C (C), and md_I (D) showing mosaic mCerulean reporter expression in the zebrafish notochord at 24 hpf. ubi:mCherry was used as injection control. N represent the number of animals expressing mCerulean in the notochord relative to the total number of animals expressing mCherry. Scale bar in (B): 0.5 mm, applies to (B, C). E, F Representative images of Ciona embryos electroporated with Monodelphis enhancer element md_C (E), and minimal forkhead promoter (fkh) only as control (F). Monodelphis enhancer element md_C expresses EGFP throughout the entire Ciona notochord, compared to minimal fkh promoter only which does not express EGFP at all (asterisk in F). N represent the number of animals expressing EGFP in the notochord relative to the total number of animals. Inserts on the top right represent bright field images of respective embryos. Scale bar in (E): 0.05 mm, applies to E, F. The species silhouettes were adapted from the PhyloPic database (www.phylopic.org).

Deletion of the three enhancer elements T3, C and I results in selective loss of Brachyury protein expression in the notochord at E9.5 and posterior defects at E12.5.

A Overview of wildtype mouse Brachyury/T/TBXTB locus adapted from the UCSC genome browser and deletion alleles generated with CRISPR-Cas9 genome editing. Exact coordinates and sequences of target sites, deletions, and genotyping primer sequences can be found in Supplementary Data 5. B–G Brachyury/T antibody staining (red) of E9.5 embryos. White dashed square in panels represents location of right bottom inserts with 2x magnification. Brachyury/T protein expression in the notochord is dose-dependent on the three enhancer elements. Asterisks in (D–G) mark absent notochord in rostral portion of the embryo. Scale bar in (B): 1 mm, applies to panels (B–G). H–M Overall morphology of E12.5 embryos with different genotypes. Blue lines indicate the location of immunofluorescence and H&E sections. Spina bifida and tail defects are dose-dependent. Arrowheads mark rudimentary tails. White lines mark spina bifida. Scale bar in H: 1 mm, applies to (H–M). N–S Dorsal view of embryos (sectioned at blue line in H–M). White lines mark areas of spina bifida. Arrowheads mark rudimentary tails compared to tails in wildtype control and double knock-out allele. Scale bar in (N): 2.5 mm, applies to panels (N–S). T–Y Immunofluorescence of mouse transverse sections. Anti-Sox2 labels the neural plate, anti-Tbxt the notochord, and DAPI marks nuclei. Sox2 expression is comparable amongst all genotypes, even in the genotypes with spina bifida, while there is loss of Brachyury/T staining in the notochord with increased loss of the enhancers. Arrowheads point to notochord. Asterisks mark absent notochord. Scale bar in (T): 0.2 mm, applies to panels (T–Y). Z–E' H&E staining of transverse sections confirm the dose-dependent loss of the notochord and spina bifida. Arrowheads point to notochord. Asterisks mark absent notochord. Scale bar in (Z): 0.2 mm, applies to (Z–E').

Bridge species establish the presence of Tbxtb enhancers across jawed vertebrates.

A Location of the enhancer elements in the human (top), gar (middle), and zebrafish (bottom) Brachyury/T/Tbxtb loci, adapted from the UCSC browser as established through the “gar bridge”. B–D Representative F0 zebrafish embryos injected with the gar enhancer elements Io_T3 (B), Io_C (C), and Io_I (D). T3 and I show mosaic mCerulean reporter expression in the notochord at 24 hpf compared to gar element C with is not active in the zebrafish notochord (asterisk). N represent the number of animals expressing mCerulean in the notochord relative to the total number of animals expressing mosaic ubi:mCherry as injection control. Scale bar in (B): 0.5 mm, applies to (B–F). E, F Representative F0 zebrafish embryos injected with the conserved zebrafish enhancer elements dr_T3 (E) and dr_I (F). T3 and I show mosaic mCerulean reporter expression in the notochord at 24 hpf. N represent the number of animals expressing mCerulean in the notochord relative to the total number of animals expressing mosaic ubi:mCherry as injection control. G, H Representative images of stable F1 embryos at 2 dpf of zebrafish enhancer elements T3 and I recapitulate the F0 expression pattern in the notochord, with dr_T3 (E) additionally expressing mCerulean in the brain, heart, and fin, and dr_I (G) in the proximal kidney close to the anal pore, pharyngeal arches, heart, fin, and spinal cord neurons. Scale bar in (G): 0.5 mm, applies to (G, H). I Phylogenetic representation of species investigated using the bridging approach with spotted gar and painted turtle as anchor species within ray-finned fish and tetrapod lineages. Arrows indicate informative phylogenetic comparisons to uncover conservation of enhancer elements T3, I, and C. The species silhouettes were adapted from the PhyloPic database (www.phylopic.org).