Myosin II and ROCK inhibition disrupts columella condensation.

<p>(A-C) Tissue slices treated with Blebbistatin (Myosin II inhibitor) or Y27632 (ROCK inhibitor). (a-d) Hoechst (blue)—nuclear DNA, Phalloidin (red)—F-actin, pSmad2 (green)—TGF-β signaling. (e) Black and white view of F-actin. (A) Normal condensation in controls with pSmad2 down regulated at HH30 (c) and formation of cell-cell actin bridges (d,e). (B,C) TGF-β signaling is maintained in explants exposed to Blebbistatin or Y27632 as indicated by high levels of pSmad2 immunolabeling (c). Condensation doesn’t occur in treated explants as indicated by the lack of an organized tissue (a) and disorganized actin (d,e). (D) Quantitative analysis indicates 2% of pSmad2 positive cells in controls, whereas pSmad2 is maintained in 80% of Blebbistatin treated cells and 68% of Y28632 treated cells (asterisk, p<0.01). Scale bars represent 75 μm (20x) and 25 μm (60x).