Fig. 3. Transgenic validation of Gli-binding regions demonstrates Gli-dependent expression in Hh target tissue. Selected candidate enhancers (Ptch1 peak2 and Nkx2.2 peak1, Rab34 peak1 and Nkx2.1 peak1) were cloned into minimal promoter lacZ reporter construct (A) to generate transgenic embryos and assayed at embryonic day 10.5. (B-G,W-Z) The embryo in panel E is cleared in benzyl alcohol and benzyl benzoate; all other whole mounts are in 80% glycerol. Histological sections through the spinal cord (forelimb level) (H-M) and brain (N-S) of transgenic and control embryos. Forelimbs (T-V,A',B') show are dissected from the corresponding whole mount. Negative control embryos and sections are shown in B,H,N,T. When compared with Ptch^lacZ/+ embryos, Ptch1 peak2-LacZ transgenics express a subset of the normal domain ß-gal activity (compare C,I,O,U with D,J,P,V), lacking expression in the limb bud mesenchyme (U,V). In situ hybridizations of Nkx2.2 (E,K,Q), transgenic Nkx2.2-lacZ embryos (F,L,R) or transgenics with the Gli-binding site mutated (G,M,S). In situ hybridizations of Rab34 (W,A') and transgenic Rab34-lacZ embryos (X,B'). In situ hybridizations of Nkx2.1 (Y) and transgenic Nkx2.1-lacZ embryo (Z). The arrows in X and Z indicate the domain of expression within the ventral diencephalon. Unlike the other transgenics, Nkx2.1-lacZ is driven by only one copy of the enhancer. All limb specimens are oriented with anterior to the left and distal up. Scale bars: 200 µm in H-S; 1 mm in A-G,T-Z,A',B'.