Figure 3. A core regulatory sequence is necessary for LacZ reporter expression in the laryngeal epithelium.

Two series of stepwise deletion constructs generated for functional dissection of MACS1, and results of the LacZ reporter assay (a). Upper left: a series of the first deletion constructs in the 807-bp DNA fragment that is sufficient for enhancer activity of MACS1 (c1, c4, c5), or in a longer 1,264-bp fragment (c2, c3). Dashed lines mark the deleted segment in each construct. Upper right: result of the transgenic assays for the deletion constructs using mouse embryos. For each construct, the fraction of embryos exhibiting reproducible reporter expression in the larynx, lung and gut relative to the total number of transgene-positive embryos at E11.5 is shown. The result indicated that the green coloured area of 29-bp is a critical region necessary for laryngeal expression. Block-m and Block-p contain the regulatory sequences necessary for expression in the lung and gut, respectively. Lower left: a second series of deletion and mutant constructs (c6 – c10) in the 807-bp fragment to delineate a core sequence (yellow coloured area) within the highly conserved 29-bp sequence (see also Supplementary Fig. 5). In the construct c11, three bases in the Fox-binding core motif are substituted from T to C (blue letters). Lower right: result of the transgenic assays for the second deletion constructs using mouse embryos. Representative reporter expression patterns in transverse sections of the transgenic embryos (b). Labels to the left of each image identify the transgenic constructs in (a). Black and white arrowheads depict presence and absence of the reporter expression specifically in the ventral laryngeal epithelium, respectively. Deletions that removed the Fox-binding core motif and its flanking sequences (c7–c9), and base substitutions in the Fox-binding core motif (c11), specifically abolished reporter expression in the laryngeal epithelia. Scale bars, 200 μm. Le, laryngeal epithelia; Lt, laryngotracheal groove.