Fig. 4. Requirement of Shh forebrain enhancers in the context of Bac 447L17ßlacZ. Schematic representation of the SBE2, SBE3 and SBE4 deletion constructs. X-gal staining in the forebrain of embryos carrying (A-D) 447L17ßlacZ^SBE2, (E-H) 447L17ßlacZ^{SBE2, SBE3}, (I-L) 447L17ßlacZ^SBE4, (M-P) 447L17ßlacZ^{SBE3, SBE4}, (Q-T) 447L17ßlacZ^{SBE2, SBE4} and (U-X) 447L17ßlacZ^{SBE2, SBE3, SBE4}. Deletion of SBE2 resulted in the loss of reporter activity at most levels of the diencephalon, including the preoptic area (POA; arrowhead in D). Compare A-D with the pattern of X-gal staining in embryos carrying the wild-type 447L17ßlacZ transgene (Fig. 3B,H,N,T). Embryos carrying 447L17ßlacZ^{SBE2, SBE3} (E-H) showed patterns of X-gal staining that were similar to those carrying 447L17ßlacZ^SBE2 (A-D). Embryos carrying 447L17ßlacZ^SBE4 (I-L) showed an absence of staining in the ventricular zone (vz) of the mge (L). In embryos carrying 447L17ßlacZ^{SBE3, SBE4} (M-P), X-gal staining was not detected in the vz or subventricular zone (svz) of the medial ganglionic eminence (mge) (P). Deletion of SBE2 and SBE4 resulted in a loss of expression in the vz of the mge and the entire diencephalon (Q-T). Embryos carrying 447L17ßlacZ^{SBE2, SBE3, SBE4} (U-X) showed no expression in the diencephalon or telecephalon. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (A,E,I,M,Q,U).