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. 2012 Aug 21;1(11):1102–1110. doi: 10.1242/bio.20122386

Fig. 3. ZPA/SHH establishes a permissive state for hairy2 expression in the distal limb defined by balanced Gli3-A/Gli3-R levels.

Fig. 3.

(Ai,ii) In situ hybridization for fgf8 and shh, revealing ZPA ablation and unaffected AER/fgf8 expression. (Aiii–vi) Distal hairy2 expression is lost upon ZPA ablation and rescued by grafting SHH-secreting QT6-SHH cells. (Bi,ii) Cyclopamine abolishes hairy2 expression in the distal limb (arrowhead). (Biii,iv) SHH-bead implantation in the AND ectopically expands hairy2 expression. Dorsal view; anterior to the top. *Bead, delimited by dashed line. (C) Immunoblots for Gli3-A, Gli3-R and β-tubulin (loading control) using total protein extracts from SHH- (circle) or FGF8- (asterisks) treated anterior limb halves and posterior FGF8-bead implanted limb halves and their contralateral controls, as schematically represented, after 6 hours of incubation. (D) Fold change in the levels of Gli3-A and Gli3-R obtained in treated tissues. FGF8 increases both Gli3-A and Gli3-R to the same extent in the posterior limb (P-FGF8), whiles it only elevates Gli3-R in the anterior domain (A-FGF8). Here, SHH greatly increases Gli3-A, down-regulating Gli3-R (A-SHH). (E) Comparison of hairy2 expression (+present; −absent) with different treatments and underlying ratio of Gli3-A/Gli3-R levels. hairy2 is expressed only when the tissue presents Gli3-A/Gli3-R>1 (brown line), defining a SHH-mediated permissive state for hairy2 expression.