Fig. 1. 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).