Fig. 4.

Fzd signaling inhibits CamkII activity. Sections of growth plate cartilage from wild-type (WT) chick embryos or from embryos infected with the indicated viruses were processed for immunofluorescence (pCamkII and/or HA in A and C) or both immunofluorescence and in situ hybridization (HA and Ihh, respectively, in B). Where indicated, DNA (blue) was stained with DAPI and actin (red) was visualized with phalloidin. (A) In the growth plate, high levels of pCamkII are normally restricted to the PHZ/HZ. White arrows indicate pCamkII signal. Infection with RCAS(A)-Wnt1 (n=6) increases cell density in the growth plate (data not shown) but does not alter the CamkII activation domain. Infection with either RCAS(A)-Wnt5a or RCAS(A)-Fzd7 decreases nuclear pCamkII levels. By contrast, inhibition of Fzd signaling by RCAS(A)-dnFzd7 infection promotes nuclear pCamkII in both the RZ and the PZ. Phalloidin staining shows that all the cells are disorganized, indicative of highly infected cartilage. n=8 for each condition. (B) Images of the PZ and PHZ (bracket) chondrocytes expressing HA-daCamkII either alone (n=5) or in combination with Wnt5a (n=5) or Wnt5b (n=5) show that Wnt signaling does not interfere with induction of Ihh by activated CamkII. Arrows indicate examples of cells expressing Ihh and brackets denote Ihh expression in the PHZ. (C) Immunofluorescence shows that co-infection with RCAS(A)-Wnt5a signaling does not block activation of endogenous CamkII (pCamkII) by RCAS(A)-HA-daCamkII (n=10). Scale bars: 50 μm in A; 200 μm in B; 100 μm in C. RZ, resting zone; PZ, proliferative zone; PHZ, prehypertrophic zone.