Fig. 1. Functional analysis of locally acting Shh enhancers in the context of Bac 429M20eGFP. Schematic of Bac clones 429M20eGFP and 389P3eGFP, which contain an eGFP insertion into Shh exon 1 and extend 180 kb upstream and 160 kb downstream of Shh, respectively. Reporter constructs carrying enhancer deletions generated in Bac 429M20eGFP are listed. (A-F) Whole-mount views of embryos carrying (A) Bac 389P3eGFP, (B) 429M20eGFP and (C-F) 429M20eGFP deletion constructs. (G-I) In embryos carrying 389P3eGFP or 429M20eGFP, strong eGFP expression was observed in the floor plate (G,I), in a pattern comparable with endogenous Shh protein (H). Embryos carrying a deletion of SFPE1 (C,K) or SFPE2 (D,L) show similar patterns of eGFP fluorescence to each other and to embryos carrying the wild type Bac (B). (J) The ventral midbrain and caudal diencephalon also show strong eGFP expression in embryos carrying 429M20eGFP and 389P3eGFP. Embryos carrying deletions of both SFPE1 and SFPE2 showed a dramatic reduction in eGFP expression in the floor plate (E,M). Embryos carrying a deletion of SBE1 showed a complete loss of eGFP staining in the ventral midbrain and caudal diencephalon (compare F,N,O with B,I,J). The absence of hindgut staining in embryos depicted in E and F is a consequence of the variable nature of the hindgut enhancer and does not reflect a dependency on sequences mediating Shh floor-plate enhancer activity. eGFP staining on sections was performed by immunohistochemistry using an anti eGFP antibody. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (A-F). di, diencephalon; fp, floor plate; mb, midbrain; nc, notochord.