Fig. 5. Phosphorylated forms of BMP-dependent R-Smads colocalize with 52 bp lacZ transgene expression in the limb and cardiac outflow tract, and are recruited to the Msx2 BMP-responsive region in native chromatin. Beads soaked in BMP4 were implanted on limbs of E11.5, 52bpMsx2-hsplacZ transgenic embryos. (A) Whole-mount lacZ stain showing expression and BMP response in the anterior limb (arrow). (B-E) Adjacent frozen sections along the dorsoventral axis of the limbs were either stained for lacZ activity (B,C; blue against Nuclear Fast Red counterstain), or immunostained with an antibody against the phosphorylated forms of the BMP R-Smads 1, 5 and 8 (D,E; pink against DAPI counterstain). (F-J) lacZ (G,I) and phospho-Smad (H,J) staining of adjacent midsagittal sections through cardiac region of a 52bpMsx2-hsplacZ embryo at E9.5 (F). A subset of phospho-Smad stained cells are positive for lacZ activity. (K-M) ChIP assay showing association of phosphorylated R-Smads with the Msx2 BMPRE in C14 limb mesenchymal cells. (K) Autoradiogram of RNA derived from BMP2-treated C14 cells, probed for Msx2. Upstream control and BMPRE primers (L) were used to interrogate chromatin immunoprecitated with anti phospo-Smad1 antibody (M).