Fig. 5. BMP2 signaling does not act as a secondary signal of Ihh to induce PTHrP expression. Forelimbs of E14.5 mouse embryos were cultured for 4 days in control medium (A,D), or in medium supplemented with Noggin (B,C), BMP2 (E), BMP2 and cyclopamine (F), or cyclopamine (G). Limbs in A,B,E-G were derived from wild-type embryos and limbs in C,D from ColII/Ihh embryos. Sections were hybridized with a riboprobe for PTHrP. (A,B) Treatment of wild-type limbs with Noggin results in a reduction of PTHrP expression (B, arrow) compared with untreated control limbs (A). (C,D) High expression of PTHrP in limbs of ColII/Ihh embryos (D) is not reduced after Noggin treatment (C). (E) BMP2-treated limbs show normal expression of PTHrP. (F,G) Cyclopamine treatment results in a block of PTHrP expression (G), which cannot be rescued by BMP2 in double treated cultures (F). In all panels, ulna is upwards and radius is downwards. cycl, cyclopamine; Nog, Noggin.