Fig. 2. Wnt3a protein re-specifies chondrogenic cells towards soft connective tissues. (A,B) Micromass cultures from chick limb mesenchyme cells treated with vehicle (A) or Wnt3a (B) for 4 days, cultured another 4 days in absence of Wnt3a. Wnt3a-treated cells failed to undergo chondrogenesis (Alcian Blue; n>50). (C) Collagen 1, tenascin C, decorin, Dermo1 and Bmp3 expression levels in high density cultures grown in continuous presence of Wnt3a (blue), or during the first 3 days of culture, after which the Wnt3a was replaced by vehicle (red). Time points 1 and 8 days were sampled twice (mean±s.e.m.). (D) Small numbers of myotubules, immunostained for myosin heavy chain (brown), accumulate in the periphery of 6-day-old mouse limb mesenchyme micromass cultures (n=6). (E) Continuous treatment with Wnt3a (250 µg/ml) slightly expands the myotubule number (n=6). (F) When Wnt3a is removed after 3 days of culture, large numbers of myotubules spread over the tissue layer (n=6). (G) Section through a chick wing 3 days after implantation of a vehicle bead at embryonic stage 22, showing the bead embedded in cartilage (n=14, Safranin O). (H) Wnt3a beads are never embedded in cartilage (n=14) but surrounded by ectopic muscle fibers, visualized by myosin heavy chain immunostaining (brown, n=7). (I,J) Section through a control chick wing (I) and a wing with implanted Wnt3a bead (J), immunostained for pro-collagen 1 (red) and myosin heavy chain (green), nuclei stained blue (DAPI) (n=4). Both sections are at a similar location and plane. Scale bars: 500 µm in A,B; 100 µm in G-J. B, bead; car, cartilage.