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. 2011 Jan 15;138(2):359–370. doi: 10.1242/dev.052324

Fig. 6.

Fig. 6.

Pthrp signaling inhibits hypertrophy by regulation of CamkII activity. (A) In situ hybridization analysis for daCamkII (infected cells) and Ihh revealed that RCAS(A)-daCamkII does not induce Ihh expression in resting chondrocytes (box) Arrows indicate ectopic expression in the proliferative zone. (B,C) To test whether Pthrp signaling antagonizes CamkII function in the resting zone, chick growth plates infected with either RCAS(A)-HA-daCamkII (green, immunofluorescence) alone (n=5), or with both RCAS(A)-Pthrp and RCAS(A)-HA-daCamkII (n=5) were analyzed for Ihh expression (red) by FISH. HA-daCamkII induced Ihh expression (white arrows) in the absence (B, left panels), but not in the presence (B, right panels), of Pthrp (white arrows). Pthrp signaling blocks activation of endogenous CamkII (pCamkII) by HA-daCamkII in the proliferative zone (C, arrows). (D) To test if Pthrp signaling acts on pCamkII through phosphatases, mouse metatarsals lacking the perichondrium were cultured in the presence of diluent (WT), 10 nM PTH1-108 (PTH) or 10 nM PTH1-108 + 2 nM calyculin A (PTH+Cal) (n=20 metatarsals per treatment). WT samples display pCamkII (red) in the prehypertrophic and hypertrophic chondrocytes (white arrow), but PTH treated metatarsals lack pCamkII (white arrow). The addition of calyculin A results in ectopic pCamkII similar to Fig. 1D. (E) Parallel experiments analyzed by western blot show similar changes in pCamkII without affecting total CamkII levels. Scale bars: 1 mm in A; 200 μm in B; 100 μm in C; 250 μm in D.