Reduced Bmp2/4 signaling in Msx1-/- ; Msx2-/- mutant AV cushions and myocardium during EMT. Panels E-H and M-P are enlarged views of the boxes in panels A-D (at E10.5) and I-L (at E11.5), respectively. Immunostaining for Bmp2/4 (green fluorescence in A, B, E, F, I, J, M and N) and phosphorylated Smad1/5/8 (red fluorescence in C, D, G, H, K, L, O and P) revealed dramatically reduced Bmp2/4-positive and phospho-Smad-positive signals in the Msx1/2 mutant AV cushion mesenchyme (red asterisks in B, F, J, N and yellow asterisks in D, H, L, P) and myocardium (white triangle arrowheads in B, D, J and L) compared with the control (A, C, E, G, I, K, M and O). On the other hand, Msx1-/-; Msx2-/- mutants exhibit broader expression patterns of both Bmp2/4 and Smad1/5/8 in the pharyngeal mesoderm compared with controls (indicated by white arrows in A-D). Q and R indicate average percentages of Bmp2/4-positive and phospho-Smad1/5/8-positive cells in the control and Msx1/2 mutant AV cushions at E10.5 and E11.5 (n = 3 for each developmental stage, P < 0.001, Student's t test). LA and LV, left atrium and ventricle; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in A (for A-D), F (for E-H), I (for I-L) and N (for M-P).