Fig. 2. A screen for long-range acting Shh regulatory sequences. Physical map displaying the distribution of genes and Bac clones spanning 1 Mb upstream of Shh. Reporter cassettes (lacZ, blue rectangle) were introduced into overlapping Bacs by random transposon insertion. (A-F) Whole-mount views of embryos stained for (A) Shh mRNA and (B-F) lacZ expression (X-gal) in representative Bac transgenic embryos harvested at 10.5 dpc. X-gal staining in the notochord was observed in embryos carrying any of three overlapping Bacs (208K5ßlacZ, 447L17ßlacZ, 265M1ßlacZ), suggesting the existence of at least two long-range acting notochord enhancers (B-D). X-gal staining in the ventral forebrain was observed in embryos carrying 447L17ßlacZ (B) and 265M1ßlacZ (D). X-gal staining in the posterior limb bud was observed in embryos carrying 265M10ßlacZ (F). None of the five embryos carrying 214O17ßlacZ exhibited reporter activity (E). di, diencephalon; fl, forelimb; hl, hindlimb; nc, notochord; tel, telencephalon. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (B-F).