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[Preprint]. 2023 Apr 20:2023.04.20.536761. [Version 1] doi: 10.1101/2023.04.20.536761

Figure 1: Human Brachyury enhancer elements T3, C and I are active in different species.

Figure 1:

(A) Human Brachyury/T/TBXTB locus with surrounding gene loci adapted from UCSC genome browser. Repeats marked in black using the RepeatMasker track. 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,C,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,F,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 cerulean 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,I,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,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.