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. Author manuscript; available in PMC: 2012 Oct 5.
Published in final edited form as: Dev Dyn. 2011 May;240(5):1212–1222. doi: 10.1002/dvdy.22634

Figure 2. An in vivo reporter assay analyzing the ZRS function in the chicken limb drives reporter gene expression in the ZPA.

Figure 2

(A) A summary of constructs tested in reporter assay. Reporter constructs containing a 1.7 kilobase fragment containing the conserved ZRS (conserved 800 base pair region represented by white rectangle) upstream of either the HSP68 (~800 base pairs), thymidine kinase (TK- ~500 base pairs), mouse minimal Shh (~1.1 kilobase), or chicken minimal Shh (~1.1 kilobase) promoters and lacZ, as well as a control construct containing the chicken minimal Shh promoter and lacZ but no enhancer sequence, were tested in an in vivo reporter assay in the chicken. The yellow rectangle in the mShh and cShh promoters represents a region of high homology in both promoters. Of these constructs, only the ZRS-cShh-lacZ construct drives reporter gene expression in the ZPA. (B, C) The HSP68-lacZ construct reproducibly drives lacZ expression throughout the entire limb bud at all stages analyzed (arrowheads point to experimental limb), including early (B, St. 20) and later (C, St. 24) stages. (D) No β-galactosidase was detectable in limbs electroporated with a control cShh-lacZ construct lacking the ZRS (n=9/9). (E-K) A high percentage of embryos electroporated with the ZRS-cShh-lacZ construct showed β-gal activity in the limb (n=43/59), and in all positively staining embryos, β-gal activity is restricted to the posterior limb in the endogenous Shh ZPA expression domain at all stages (St.) analyzed, including St. 20 (E, I), St. 21 (F, J), St. 22 (G), St. 26 (H, K). Also shown is Shh mRNA expression for comparison (L). In I-L, posterior is on the bottom.