Normal proliferation but decreased total cell numbers in Msx1-/- ; Msx2-/- mutant AV cushions during EMT. Immunostaining with the anti-BrdU antibody at both E11.5 and E12.5 revealed comparable levels of cell proliferation in the AV cushions and atrial and ventricular myocardium of Msx1-/-; Msx2-/- mutants and their littermate controls (compare A with B and E with F). Immunostaining against α-smooth muscle actin (α-SMA) showed dramatically reduced numbers of α-SMA-positive mesenchymal cells in the Msx1/2 null mutant AV cushions compared with the control (red arrows in D and H indicate sparse α-SMA-positive cells in the double mutant AV cushions). White asterisks in A, B, E and F, and red asterisks in C, D, G and H indicate the location of the AV cushions. I, Statistical analyses revealed significantly reduced total cell numbers in the AV cushions of the Msx1/2 double mutants compared with those of their littermate controls (at least 4 sections were counted for each embryo, and 3 pairs of Msx1/2 double mutants and their littermate controls were analysed; P < 0.01 for both E10.5 and E11.5, Student's t test). IVS, interventricular septum; LA and LV, left atrium and ventricle; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in A (for A, B), C (for C, D), F (for E, F) and H (for G, H).