Fig. 6. The 5’ 190 bp enhancer is necessary for Bmp6 expression.

(A) Schematic of the genomic location of the 180 kb CHORI BAC29E12 with respect to Bmp6 and nearby genes (coding regions shown in black are Ipo4, Pdcd6, Txndc5, Muted, Eef1e1, and Slc35b3 from left to right). (B) Recombineering strategy for introducing GFP into the first exon of Bmp6; grey bars indicate exons. (C) Final circular BAC with inverted Tol2 sites for transposition and GFP reporter (not to scale). (D) Strategy for introducing TALEN lesions into the 190 bp 5’ enhancer. The same TALENs were used to target the enhancer in stable transgenic BAC fish and at the endogenous Bmp6 locus (diagram not to scale). (E) Sequences of stable mutant enhancer alleles. For the endogenous locus targeting, F2 fish trans-heterozygous for two different enhancer mutations were generated. Fish in (M) carried alleles 1 and 2; fish in (O) and (Q) carried alleles 1 and 3. The predicted Smad3 binding site is indicated with blue text in the wild type sequence. (F, G) In the reporter BAC, TALEN injection frequently severely reduced GFP expression from the pectoral fin relative to controls at 5 dpf. A small patch of mosaic, unaffected GFP is indicated with the arrow in (G). (H, I) TALEN injection also eliminated much of the Bmp6 tooth expression (I). (J, K) GFP expression was also reduced in gills (asterisk) and slightly reduced in the gill rakers (arrowhead). (L–M). Mutations in the enhancer caused a reduction in pectoral fin expression relative to wild-type siblings. (N, O) Bmp6 expression was also lost in tooth epithelia (arrows), but was not entirely lost in mesenchyme (arrowheads). (P, Q) Expression was also noticeably reduced in gills (asterisk), though gill raker expression (arrows) appears similar to wild-type sibling controls. Scale bars = 100 µm.