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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.
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