Fig. 4

Pharmacological disruption of TGFβ signaling or TALEN-induced mutations of the predicted Smad3 binding site reduce enhancer activity. (A–C) Treatment of stickleback fry for 7 days in SB431542 (an ALK5 inhibitor) severely reduced GFP expression driven by the 190 bp enhancer in a dose-dependent manner. Expression was severely reduced in the epithelia (arrows), but not mesenchyme (asterisks), of pharyngeal teeth at both low (25 µM, B) and high (50 µM, C) doses relative to controls (A). (D-F) SB431542 also eliminated GFP driven by the stickleback enhancer in a zebrafish trans environment. (G) The sequence targeted by TALENs contains a predicted Smad3 homodimer binding site (blue). The TALEN binding sites are indicated in purple text and the purple scissors indicate the approximate site of endonuclease activity. The XbaI site used for molecular screening is underlined in green, and the mutagenized sequence of the Smad3 binding site, indicated by orange letters, is shown below. (H-I) Injection of the TALENs into stable transgenic fish carrying the 190 bp reporter construct resulted in near complete loss of GFP expression in 95% of injected animals (I) relative to controls (H). Residual GFP seen in (I) is likely the result of the mosaicism of TALEN-induced lesions. (J). Mutating the predicted Smad3 binding site resulted in a loss of GFP expression in both epithelium and mesenchyme of pharyngeal teeth in 3/3 stickleback lines observed. Bone is fluorescently counterstained with Alizarin red. Scale bars=200 µm.