Fig. 3. Wnt and FGF proteins act in synergy to promote proliferation and maintain the undifferentiated state. (A-G) Alcian Blue staining. (A) Fgf8 protein delays chondrogenesis in micromass cultures of chick stage 22-23 limb mesenchyme (n=12). (B) Wnt3a combined with Fgf8 blocks chondrogenesis (n=12). (C) Removal of Fgf8 at day 4 of culture has little effect on chondrogenesis (n=12). (D) Cells treated with Wnt3a and Fgf8 resume chondrogenesis upon removal of the factors at day 4 (n=12). (E-G) Limb mesenchyme cells were expanded for 4 days in presence of Wnt3a alone (E) or in combination with Fgf8 (F,G), trypsinized and replated as micromass cultures. Cells expanded in Wnt3a alone lost their chondrogenic potential (E). Cells expanded in Wnt3a and Fgf8 retained their chondrogenic potential (F), whereas Wnt3a was still able to inhibit their chondrogenesis (G) (n=4). (H) Wnt3a promotes proliferation of limb mesenchyme in micromass cultures, which is enhanced by Fgf8. Fgf8 alone does not enhance proliferation (n=4). (I) Size of limbs cultured 4 days with intact ectoderm, or without ectoderm in presence of the indicated factors (n=8, mean±s.e.m.). (J-N) Representative examples of Alcian Blue-stained limb buds cultured without ectoderm in presence of vehicle (J), Wnt3a (K), Fgf8 (L), Wnt3a and Fgf8 (M), or with ectoderm left intact (N). Scale bar: 500 µm in J-N.