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Fig. 4. Ectopic expression of chick Pcl2 suppresses Shh expression in the ZPA of the chick and mouse limb buds and in the chick feather buds. (A) Chick Pcl2 transcripts are detected in the medial mesenchyme of a stage 18 chick wing bud. The anteroposterior orientation is indicated. (B) A stage 22 chick embryo shows complete repression of Shh expression in the ZPA (red arrow) of the right wing infected with RCAS-Pcl2 and normal Shh expression in the control wing (black arrow). (C,D) Chick Pcl2 expression is detected in the mesenchyme of a feather bud from a stage 31 chick embryo (C), while Shh expression is restricted to the epithelium of a feather bud from the same stage (D), exhibiting a complementary expression pattern to that of chick Pcl2. (E) Shh expression is inhibited in feather buds (arrows) infected with RCAS-chick Pcl2. There is normal Shh expression (arrowheads) in adjacent uninfected feather buds. (F,G) Chick Pcl2 transgene expression (arrow) is detected in a hindlimb of an E10.5 transgenic embryo (G), but not in a wild-type limb (F). (H,I) Transgenic expression of chick Pcl2 in the mesenchyme of mouse developing hindlimb suppresses Shh expression in the ZPA (arrow in I), when compared with a control hindlimb from E10.5 nontransgenic littermate (H). Scale bars: 100 µm in A; and 50 µm in C,D.

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