BMP inhibition results in dorsal to ventral transformation of columella condensation morphology.

<p>(A,B) Application of the BMP inhibitor LDN193189. (a-c) Hoechst (blue)—nuclear DNA, Phalloidin (red)—F-actin, pSmad2 (green)—TGF-β signaling. (d) Black and white view of F-actin. (C,D) Graphs comparing percentage of pSmad2 positive cells and cell density in controls and LDN193189 treated tissue. (E) <i>COL2A1</i> in situ hybridization in LDN193189 and SU5402 treated tissue. (F) Phalloidin immunolabeling of dorsal and ventral cartilages of the second pharyngeal arch. (A,C) pSmad2 is detected at HH30 in 3% of controls. (B,C) Where BMP is inhibited, TGF-β signaling is maintained in a significant number of cells (47%, asterisk, p<0.01). (d) Tissue exposed to LDN193189 exhibits morphology that is reminiscent of the ventrally situated extracolumella. (D) Similar to extracolumella cell counts, LDN193189 treated tissue has an increased cell density compared to controls (asterisk, p<0.01). (E) <i>COL2A1</i> expression is unaffected by the inhibitors LDN193189 (BMP) and SU5402 (FGF) indicating no effect on overt differentiation of chondrocytes. (F) Dorsal (otic capsule) and ventral (ceratorbranchial and epibranchial) cartilage have distinct actin morphology, irrespective of their fate as replacement or persistent cartilage. Scale bars represent 150 μm (10x), 75 μm (20x) and 25 μm (60x).