Fig. 4. BMP2 does not rescue the advanced onset of hypertrophic differentiation induced by a loss of Ihh signaling. Forelimbs of E14.5 embryos were cultured for 4 days in control medium (A,B) or in medium supplemented with PTHrP (C,D), PTHrP and cyclopamine (E,F), cyclopamine (G,H), BMP2 and cyclopamine (I,J) or BMP2 (K,L). Serial sections were hybridized with riboprobes for Ihh (A,C,E,G,I,K) or ColX (B,D,F,H,J,L). (A-D) Treatment with PTHrP results in a delay of hypertrophic differentiation, as seen by the reduced expression of Ihh (A,C) and ColX (B,D), and the increased distance between the Ihh expression domain and the joint region. Treatment with cyclopamine results in an advanced onset of hypertrophic differentiation as can been seen by the enlarged domain of Ihh and ColX expression (G,H). (E,F) PTHrP can rescue the advanced onset of hypertrophic differentiation in explants co-treated with PTHrP and cyclopamine. (I,J) Co-treatment with BMP2 and cyclopamine does not rescue the advanced onset of hypertrophic differentiation induced by cyclopamine, but leads to a further enlargement of the Ihh expression domain (I) compared with limbs treated with either cyclopamine (G) or BMP2 (K). Limbs treated with cyclopamine and BMP2 plus cyclopamine were derived from the same embryo. In all panels ulna is upwards and radius is downwards. cycl, cyclopamine.
