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Fig. 1. Limb ectoderm inhibits chondrogenesis and promotes proliferation via Wnt signals. (A) Chick limb ectoderm cultured on top of E11.5 Axin2^lacZ/+ limb mesoderm cells induces the reporter in the mesoderm (n=10). (B) Chick limb ectoderm cultured on top of chick limb mesoderm inhibits chondrogenesis (stained with Alcian Blue) in a 150 µm wide zone around the explant (n=30). (C) The Wnt antagonist Fz8CRD inhibits induction of the Axin2^lacZ/+ reporter by the ectoderm (n=10). The ectoderm in A and C is derived from stage 24 chick limb buds and does not carry the reporter. (D) Fz8CRD protein abrogates the chondro-inhibitory effect of the ectoderm (45 out of 48). (E,F) E11.5 Axin2^lacZ/+ limb bud cells induce the lacZ reporter (blue) in response to Wnt3a protein (100 ng/ml for 19 hours; F). (G,H) Chick stage 22 limb mesenchyme cells undergo chondrogenesis (stained with Alcian Blue) in micromass cultures (G), which is inhibited by 100 ng/ml Wnt3a protein (H, n>200). (I,J) Activation of the reporter by Wnt3a beads (arrowheads, J) but not vehicle beads (I) implanted in E11.5 Axin2^lacZ/+ limb buds (n=4). (K,L) Vehicle beads implanted in stage 22 wing buds become embedded in the cartilage of the humerus (K), whereas chondrogenesis (stained with Alcian Blue) is inhibited around Wnt3a beads (L, n=8). (M,N) Chick limb ectoderm cultured on top of chick limb mesoderm induces BrdU incorporation (blue) in the surrounding cells (M, n=17), which is inhibited by Fz8CRD (N, n=18; nuclei labeled in red). (O,P) Sections through limb buds cultured with implanted vehicle (O) or Wnt3a (P) beads. Wnt3a induces BrdU incorporation around the bead (n=11). Scale bars: 100 µm in A-D,M-P; 500 µm in E-H; 200 µm in I-L. h, humerus; r, radius; u, ulna; B, bead; E, ectoderm.

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