A 190 base pair, TGF-β responsive tooth and fin enhancer is required for stickleback Bmp6 expression

• A 190 bp enhancer of Bmp6 drives expression in developing fins and teeth.
• Enhancer function has been conserved for >250 million years of teleost evolution.
• This enhancer requires a predicted Smad3 binding site.
• This enhancer is turned off by pharmacological disruption of TGF-B signaling.
• This enhancer is required for expression of Bmp6 in fins and teeth.

The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190 bp enhancer ~2.5 kilobases 5′ of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72 bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFβ effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.