Extended Data Fig. 4. The holostean bridge connects vertebrate Tbx4 gene enhancers.

(a) Human fetal TBX4 chromatin accessibility shows a peak at the ‘lung mesenchyme enhancer’ (LME) in lung cells. (b) Human TBX4 single cell expression in fetal lung. See Supplementary Note 9 for details. (c) VISTA SLAGAN alignments with human (top), bowfin (middle), and stickleback (bottom) as reference. Top: Human TBX4 intron 3 ‘lung mesenchyme enhancer’ (LME, red box), driving expression in the developing mammalian lung bud^60,61, shows conservation with other tetrapods, coelacanth, bowfin, and gar (and also with bichir^12,126 and some sharks¹²⁷, not shown), but no conservation with teleosts. HLEB: TBX4 hind limb enhancer B⁶⁰ (black box). Bottom: The stickleback tbx4 gene contains three putative ‘swimbladder enhancers’⁸⁶ (SBE1-3, green boxes). SBE2 is conserved with bowfin but not lobe-finned vertebrates. Intron 3 regions R1 and R2 (blue boxes) appear as conserved among neopterygians (teleosts, bowfin, gar), but not with coelacanth or tetrapods. HLEB (black box) is also present. Middle: The bowfin tbx4 gene identifies different conserved regions defined in the human- and stickleback-centric alignments (top, bottom): SBE2 (green box), R2, and HLEB. Lobe-finned LME and neopterygian R1 elements overlap within the bowfin genome, and are thus orthologous conserved non-coding elements, connected via the holosteans bridge (right). This bowfin LME/R1 region is also a bowfin developmental ncOCR (main text Fig. 4f, where it is shown on the reverse strand).